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Differential plant responses, morphometrics and electrophoretic patterns of some Meloidogyne spp. from Costa Rica and Florida, U.S.A., and the description of Meloidogyne salasi sp. n.

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Title:
Differential plant responses, morphometrics and electrophoretic patterns of some Meloidogyne spp. from Costa Rica and Florida, U.S.A., and the description of Meloidogyne salasi sp. n.
Creator:
Lopez Chaves, Roger, 1945-
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English
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x, 124 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Enzymes ( jstor )
Female animals ( jstor )
Gels ( jstor )
Head ( jstor )
Juveniles ( jstor )
Rice ( jstor )
Root knot nematodes ( jstor )
Roundworms ( jstor )
Species ( jstor )
Tomatoes ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Meloidogyne ( lcsh )
Plant nematodes -- Florida ( lcsh )
Root-knot ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1984.
Bibliography:
Bibliography: leaves 114-123.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Roger Lopez Chaves.

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DIFFERENTIAL PLANT RESPONSES, MORPHOMETRICS AND ELECTROPHORETIC PATTERNS OF SOME Meloidogyne spp. FROM COSTA RICA AND FLORIDA,
U.S.A., AND THE DESCRIPTION OF Meloidogyne salasi sp. n.




By

ROGER LOPEZ CHAVES


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1984















This dissertation is dedicated first of all to the gentleman who

inspired me in the study of the science of nematology, the distinguished Costa Rican scientist Professor Luis Angel Salas Fonseca. Thank you, Don Luis, for letting me drink from the stream of your vast knowledge for so many years, and for giving me the opportunity to meet a living example of a wonderful human being.

The work is also dedicated to my parents, R6ger L6pez C. and

Francisca Chaves M., to my wife Ana I. Chaverri M. and to our children, Susana Maria, Roberto Enrique, Jos& Francisco and Juan Pablo.















ACKNOWLEDGMENTS

The facilities provided by Dr. D.W. Dickson, chairman of the supervisory committee, are greatly appreciated.

Gratitude is extended to Dr. G.C. Smart, Jr., Dr. R.E. Stall, Dr. R.A. Dunn, and Dr. J.R. Rich for serving as members of the supervisory committee.

The help provided by Mr. F.E. Woods and Mr. R.A. Henn in reviewing and correcting the first manuscript, and Mr. A.L. Taylor with the photographic work is acknowledged also.

Special thanks are given to my colleague, Ing. Agr. Luis Alejandro Salazar Figueroa, for his invaluable help during several years, and to all the personnel at the Laboratorio de Nematologia, Facultad de Agronomia, Universidad de Costa Rica.

The patience and understanding of my wife Ana Isabel and our children were vital for the success of this investigation.

The financial support of the Consejo Nacional de Investigaciones Cientificas y Tecnol6gicas is deeply appreciated, as well as the assistance provided by the Universidad de Costa Rica.
















TABLE OF CONTENTS
Page

DEDICATION....................................................... ii

ACKNOWLEDGMENTS .................................................. iii

LIST OF TABLES..................................................... v..v

LIST OF FIGURES................................................... vii

ABSTRACT........................................................... ix

CHAPTER

I INTRODUCTION............................................... 1

II VARIABILITY OF Meloidogyne spp. FROM COSTA RICA......... 4
Introduction........................................... 4
Materials and Methods................................. 11
Results............................................... 16
Discussion............................................ 30

III DESCRIPTION OF Meloidogyne salasi sp. n................. 37
Introduction.......................................... 37
Materials and Methods................................. 38
Species Description................................... 40

IV VARIABILITY OF Meloidogyne incognita FROM FLORIDA....... 73
Introduction.......................................... 73
Materials and Methods................................. 77
Results............................................. 80
Discussion............................ ............ 88

V ELECTROPHORETIC PATTERNS OF SOME Meloidogyne spp. FROM
COSTA RICA AND FLORIDA.................................. 92
Introduction......................................... 92
Materials and Methods................................. 93
Results ........*****..................... .................. 97
Discussion............................................ 101

VI CONCLUSIONS ............................................. 104

APPENDIX ..........**********....................................... .108

LITERATURE CITED ..............** * **.................... ..................114

BIOGRAPHICAL SKETCH .............................................. 124















LIST OF TABLES


Table e

1 Designations, sources and selected ecological characteristics of the collection sites of 16 populations of
Meloidogyne spp. from Costa Rica......................... 13

2 Interpretation of the predominant type of perineal pattern
of females of 16 populations of Meloidogyne spp. from Costa
Rica ..................................................... 17

3 Morphometric characters of females of 16 populations of
Meloidogyne spp. from Costa Rica......................... 22

4 Morphometric characters of second-stage juveniles of 16
populations of Meloidogyne spp. from Costa Rica.......... 24

5 Morphological characters of males of 16 populations of
Meloidogyne spp. from Costa Rica......................... 27

6 Response of seven differential plants to 16 populations of
Meloidogyne spp. from Costa Rica......................... 29

7 Measurements of 50 females and eggs of Meloidogyne salasi
sp. n. from rice, cv. C.R.1113........................... 41

8 Measurements of 50 males of Meloidogyne salasi sp. n. from
rice, cv. C.R.1113....................................... 55

9 Measurements of 50 second-stage juveniles of Meloidogyne
salasi sp. n. from rice, cv. C.R.1113.................... 63

10 Response of seven differential plants to three populations of Meloidogyne incognita from Florida.................... 81

11 Interpretation of the predominant type of perineal pattern of females of three host races of Meloidogyne
incognita from Florida................................... 82

12 Comparative morphological data (im) from females of three host races of Meloidogyne incognita from Florida......... 85

13 Comparative morphological data (pm) from second-stage juveniles of three host races of Meloidogyne incognita
from Florida ............................................. 86

14 Comparative morphological data (pim) from males of three host races of Meloidogyne incognita from Florida.............. 87










Table Page

15 Range of measurements of females from 16 populations of Meloidogyne spp. from Costa Rica......................... 108

16 Range of measurements of males from 16 populations of Meloidogyne spp. from Costa Rica......................... 109

17 Range of measurements of infective second-stage juveniles from 16 populations of Meloidogyne spp. from Costa Rica.. 110 18 Range of measurements of females from three host races of Meloidogyne incognita from Florida....................... 111

19 Range of measurements of second-stage juveniles from three host races of Meloidogyne incognita from Florida......... 112 20 Range of measurements of males from three host races of
Meloidogyne incognita from Florida....................... 113















LIST OF FIGURES


Figure Page

1 Map of Costa Rica showing the approximate location of
the collection sites of 16 populations of Meloidogyne
spp ...................................................... 12

2 Photomicrographs of female perineal patterns of nine
populations of Meloidogyne spp. from Costa Rica.
CR4: M. arenaria. CR1, 3, 5, 6, 11, 12, 16, and
17: M. incognita ......................................... 20

3 Photomicrographs of female perineal patterns of six
populations of Meloidogyne spp. from Costa Rica.
CR2: M. sp.. CR7, 9: M. exigua. CR10, 14, and
15: M. hapla ............................................. 21

4 Outlines of females of Meloidogyne salasi sp. n.......... 43

5 Female of Meloidogyne salasi sp. n....................... 44

6 Scanning electron photomicrographs of face views of
females of Meloidogyne salasi sp. n...................... 46

7 Cephalic region of a female of Meloidogyne salasi sp. n.. 48

8 Anterior region of a female Meloidogyne salasi sp. n..... 49

9 Photomicrographs of female perineal patterns of Meloidogyne
salasi sp. n.. A, B and C from Costa Rica. D from Panama. 51

10 Perineal patterns of Meloidogyne salasi sp. n.. A, B and C
from Costa Rica. D from Panama ......................... 52

11 Scanning electron photomicrographs of female perineal
pattern of Meloidogyne salasi sp. n...................... 53

12 Males of Meloidogyne salasi sp. n.. A Esophageal region
(ventral). B Cephalic region (lateral). C,D Tail
(lateral) ................................................ 56

13 Anterior region of males of Meloidogyne salasi sp. n.. A,B
Scanning electron microscope photomicrographs. C,D Light
microscope photomicrographs.............................. 58

14 Scanning electron photomicrographs of males of Meloidogyne
salasi sp. n.. A,B Face views. C Lateral field.
D Tip of spicules showing pores.......................... 60

vii










Figure Page

15 Second-stage juveniles of Meloidogyne salasi sp. n..
A Esophageal region (lateral). B Cephalic region
(lateral). C Tail (dorsal). D Tail (lateral)............ 64

16 Scanning electron photomicrographs of face views of secondstage juveniles of Meloidogyne salasi sp. n............... 65

17 Lateral field of second-stage juvenile of Meloidogyne salasi
sp. n..................................................... 66

18 Second-stage juveniles of Meloidogyne salasi sp. n.. A,B
Anterior region. C,D Tail terminus....................... 68

19 Photomicrographs of female perineal patterns of three
populations of Meloidogyne incognita from Florida......... 83

20 Diagramatic sketch of comparative electrophoretic patterns
of some Meloidogyne spp. from Costa Rica and Florida.
Left to right: M. salasi sp. n.; M. exigua (CR7); M. exigua (CR9); M. hapla (CR10); M. hapla (CR14);
M. arenaria; M. incognita (CR3); M. incognita (CR11);
M. incognita (CR12); M. incognita (CR16); M. incognita
(M-195); M. incognita (M-165) and M. incognita (M-198).... 98


viii















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


DIFFERENTIAL PLANT RESPONSES, MORPHOMETRICS AND ELECTROPHORETIC
PATTERNS OF SOME Meloidogyne spp. FROM COSTA RICA AND FLORIDA,
U.S.A., AND THE DESCRIPTION OF Meloidogyne salasi sp. n.

By

R6ger L6pez Chaves

August 1984

Chairman: D.W. Dickson
Major Department: Entomology and Nematology

Based on a morphometric study of males, females and second-stage juveniles, and on the responses of seven differential plants, five species of Meloidogyne were distinguished among 16 populations collected at different locations in Costa Rica. These were M. arenaria, M. incognita, M. hapla, M. exigua and an undescribed species, found infecting rice. The responses of the differential plants indicated that the M. arenaria population was host race 2 (does not infect peanut) and that the M. incognita populations included host races 1 and 2.

Evidence of pathogenic variation was found between two M. exigua

populations. One reproduced readily on tomato, whereas the second population did not. Similarly, two populations of M. hapla reproduced readily on pepper, whereas the third population reproduced only to a limited extent on that host.

Three populations of M. incognita from Florida, U.S.A., were distinguished as host races 1, 2, and 3 based on morphometrics and









differential plant responses. These were compared to ten populations of Meloidogyne spp. from Costa Rica by means of starch gel electrophoresis. With a few exceptions, malate dehydrogenase, phosphoglucose isomerase, fumerase, a-glycerophosphate dehydrogenase, and isocitrate dehydrogenase isozyme patterns could be used to differentiate the species of Meloidogyne that were investigated. Intraspecific differences were also noted in patterns of the five enzymes between the two populations of M. hapla and in the patterns of all enzymes except isocitrate dehyrogenase between the two populations of M. exigua.

Meloidogyne salasi sp. n., a pathogen of rice (Oryza sativa L.) in Costa Rica and Panama, was described and illustrated. It can be distinguished from related species (M. kralli, M. acronea and M. graminis) by the areolation of the lateral fields in the male, the dimensions and characters of the perineal pattern of the females, and by the total length and a, b, and tail length/anal width ratios of the infective second-stage juveniles.
















CHAPTER I
INTRODUCTION

The broad geographical distribution, wide host range, severe

pathogenic effects and synergistic interactions with many other kinds of plant disease organisms, have placed root-knot nematodes (Meloidogyne Goeldi, 1887, Nematoda: Meloidogynidae) among the major plant pathogens affecting man's food supply (Taylor and Sasser, 1978; Sasser, 1980; Sasser and Carter, 1982). Practically every crop grown is infected by one or more species of this genus. Not only are yields affected but quality is also reduced, particularly in the case of root crops.

Management strategies aimed at reducing the severity of the damage caused by Meloidogyne spp. include the use of chemicals, crop rotation, resistant cultivars and other cultural practices (Taylor and Sasser, 1978; Sasser and Carter, 1982). The last three tactics require extensive knowledge of the morphology, variability and ecology of the species causing the damage.

One of the problems associated with the implementation of nonchemical management tactics against root-knot nematodes is the correct identification of populations. Identification is complicated by the variation in morphology and host range commonly present in species of this genus (Netscher, 1978; Whitehead, 1968). Due to this variability, approaches other than classical morphology have been used to identify species. Among those are differential host test (differential plants)










(Taylor and Sasser, 1978), biochemical analysis of different enzymatic and nonenzymatic proteins (Hussey, 1982), cytology, and mode of reproduction (Triantaphyllou, 1982).

The identification and/or quantification of the variability within and among species of root-knot nematodes by these different approaches could provide the basis for a better understanding of the genus, not only from the morphological, but from the physiological and ecological points of view as well. This understanding would enable recognition of those characters which are species specific and therefore reliable for distinguishing species, as well as recognition of characters with little or no value in the identification of field populations due to their overlap among or between species or their instability. Eventually, this could lead to the development of a faster, more accurate methodology for the identification of species and/or races within a species. We would then have a better basis for the planning and implementation, both locally and internationally, of management strategies for this important group of plant pathogens.

Research was initiated in late 1980 with four objectives: a) to study the variability of some populations of root-knot nematodes from Costa Rica by a morphometric characterization of males, females, and infective second-stage juveniles, and by their reaction on certain differential plants; b) to characterize three populations of M. incognita (Kofoid and White, 1919) Chitwood, 1949 from Florida, U.S.A., by morphometrics and the responses of differential plants, and to compare them to populations of this same species from Costa Rica; c) to use starch gel electrophoresis to differentiate several species of root-knot nematodes







3


found in Costa Rica, compare populations of M. incognita from both Costa Rica and Florida, and investigate possible differences among the latter; and d) to describe and illustrate a new species of root-knot nematode found infecting rice in Costa Rica and Panama.















CHAPTER II
VARIABILITY OF Meloidogyne spp FROM COSTA RICA Introduction

Papilla and Yuquilla, meaning small potato and small cassava, are common names applied by some farmers in Costa Rica to the root-knot disease of numerous cultivated plants. Others simply call the disease Nematodos or Meloidogyne.

Until recently, the reports of root-knot nematodes in Costa Rica

were scattered, the first one apparently being that of von Bulow (1934). This author reported "vermes of the genus Heterodera" inside abnormal soybean nodules and in galled peach roots. A year later Heterodera was again found on peaches in San Pedro de Montes de Oca (von Bulow, 1936). By 1935 root-knot nematodes were found in melon, cabbage and tobacco, in addition to coffee and Inga (von Bulow, 1935). Some observations were published on the occurrence of nematodes on Inga and coffee collected from several locations in the Central Plateau (von Bulow, 1937). Many of the die-back problems in coffee were attributed to the infection by nematodes. His illustrations of root galls on both plant species, as well as those of nematode eggs, juveniles and females, seemed to correspond to a species of the genus Meloidogyne, as pointed out previously by Salas and Echandi (1961).

Sixteen years later M. incognita var. acrita Chitwood, 1949, was identified for the first time in Costa Rica (Taylor and Loegering, 1953). These authors reported a low incidence of this species in abaca.









Olsen and Thomas (1954) successfully controlled M. incognita var. acrita on tomato and okra with DD and EDB. They also found aldrin and parathion did not give satisfactory results.

A few years later Van der Laat (1960) successfully controlled M.

incognita on tomato with DBCP, 1,3-D and DD in a sandy loam soil but not in a clay loam.

More recently, Ramirez (1971) did not obtain yield increases in

tomato with the application of ethoprop at 10 and 20 kg ai/ha, but there were reductions in the number of nematodes in the soil and in the rootknot index up to three months after transplanting. He found that fensulfothion gave higher yields than ethoprop at the same rates, although its effect on M. incognita was not as severe as that of ethoprop.

Salas and Echandi (1961) demonstrated the pathogenicity of M.

exigua Goeldi, 1887, on coffee seedlings. They mentioned that under field conditions plants infected by this nematode showed above ground symptoms of wilting, chlorosis, defoliation and low yields, whereas below ground galls appeared mostly on the finer roots. They considered this nematode induced a serious disease of coffee.

In 1968 Figueroa (1973) found a species of root-knot nematode

infecting rice in Volcan de Buenos Aires, Puntarenas. He later studied its life cycle and illustrated some of its morphological characters. He also demonstrated its pathogenicity on 12 rice genotypes.

Salas (1975) mentioned the presence of M. incognita in the Atlantic zone, the Central Plateau and the high mountains of Costa Rica. He also reported that M. exigua, M. hapla Chitwood, 1949, and M. javanica (Treub, 1885) Chitwood, 1949, were common in the Central Plateau.









Another report about Meloidogyne in Costa Rica is that of Pessoa (1973). He found root-knot nematode juveniles in virgin soil in the Atlantic zone that was to be planted with bananas.

Recent surveys (Gonzalez, 1978a; Gonzalez, 1978b; Gonzalez, 1979; Lopez, 1978; Lopez, 1980c; Lopez and Azofeifa, 1980; Lopez and Azofeifa, 1981; Lopez and Salazar, 1978; Lopez et al., 1980) and collections of field populations (Alvarado and Lopez, 1982; Hidalgo and Lopez, 1980a; Salazar, 1980a; Salazar and Lopez, 1980) further showed the wide host range and widespread occurrence of root-knot nematodes throughout the country. The species mentioned were M. hapla, M. exigua and M. javanica. An undescribed species was also found infecting rice in the southeastern part of the country (Alvarado and Lopez, 1981; Lopez, 1981a; Sancho, 1981). Numerous crops and weeds were cited as hosts of the different species of root-knot nematodes, including some new ones (Lopez, 1980c; Lopez and Salazar, 1978).

The pathogenicity of these species of root-knot nematodes on several crops is beginning to be studied. Results of experiments carried out with M. incognita on lettuce (Castro and Lopez, 1981; Gonzalez and Lopez, 1980b), common bean (Lopez, 1980b), corn (Hidalgo and Lopez, 1980b) and with the undescribed species on rice (Sancho, 1981) showed that rootknot nematodes can significantly reduce the growth and/or yield of these crops. These losses can be even higher if the nematodes interact with other plant pathogens, as was shown with Fusarium oxysporum f. sp. pisi and M. incognita plus M. hapla on green peas (Padilla et al., 1980).

Indirect evidence of the damage root-knot nematodes can inflict on both field and vegetable crops was obtained with the experimental use of nematicides and other management practices. In air-cured tobacco, the









application of DD increased yield by 20% (Lopez and Fonseca, 1978), whereas materials such as ethoprop and carbofuran increased yields of burley and flue-cured tobacco by 9 and 10%, respectively (Calvo and Lopez, 1980; Carrillo and Lopez, 1979). Greater increases were obtained in certain vegetable crops. For instance, metham-sodium, phenamiphos, carbofuran and aldicarb increased carrot yields 238, 135, 114, and 72%, respectively over the untreated controls (Perlaza et al., 1979). In lettuce, the same chemicals increased yields by 112, 219, 117, and 91%, respectively over the control (Perlaza et al., 1978). Both of these experiments were carried out under high initial population densities. However, when the initial population density was relatively low for lettuce, the increase was only 13% with phenamiphos (Mattey and Lopez, 1978).

Some nonfumigant nematicides have also given satisfactory results on green peas (Padilla and Lopez, 1979). Yield increases of 35, 21, 18, and 27% were obtained with aldicarb, two commercial formulations of carbofuran and with ethoprop, respectively. Use of phenamiphos or fensulfothion significantly reduced the root-knot index and the density of juveniles in the soil at harvest time, but failed to increase the yield. It appeared that these two chemicals may be phytotoxic to green peas.

In celery, the elimination of the previous crop residue (roots) severely infected with M. incognita increased yields by only 3%. The incorporation of organic matter (a mixture of broiler manure and sawdust) caused a 13% decrease in celery weight and promoted significantly higher root-knot indices 46 and 94 days after transplanting. The application of aldicarb did not affect celery yield (Incer and Lopez, 1979). In a









subsequent study (Rivera and Lopez, 1982), carbofuran, phenamiphos and aldicarb increased the yield of the celery cultivar 'Dwarf', but these materials failed to produce higher yields with the cultivar '5205'. Ethoprop and phenamiphos apparently had a phytotoxic effect.

In corn, no significant differences in nematode populations at harvest time or yield were found between carbofuran, fensulfothion, phenamiphos and ethoprop and the untreated control (Gonzalez and Lopez, 1980a).

A few morphological and morphometric studies of some populations of root-knot nematodes were conducted in the recent past (Hidalgo and Lopez, 1980a; Lopez and Salazar, 1978; Salazar, 1980a; Salazar and Lopez, 1980). M. hapla was found widespread in the Central Volcanic Range, at altitudes between 1,360 and 2,501 m above sea level. This area has average temperatures between 14 and 18*C, with precipitation over 2,000 mm per year. The soil type is Andept. In most cases the range and average values of several characters of second stage juveniles, and of certain characters of the perineal pattern, were similar to those reported previously for this species, although some discrepancies were found. For instance, the striae of the perineal pattern were mostly unbroken, in contrast with the report by Esser et al. (1976), and a dilated rectum was observed in some second-stage juveniles. In both this and another study (Salazar and Lopez, 1980) with second stage juveniles of M. hapla, significant differences among populations were found in the distance between the dorsal esophageal gland orifice and the base of the stylet knobs (DEGO), tail length, maximum body width, anal width and the a ratio. Differences among populations were also









noted in the c ratio (Salazar and Lopez, 1980) and in the body length (Lopez and Salazar, 1978).

A similar situation was found for M. incognita; i.e., the range and mean values for females, males and second-stage juveniles were similar to those reported earlier for this species, but some significant differences for certain characters were found among populations (Hidalgo and Lopez, 1980a; Salazar and Lopez, 1980). This species is the most prevalent one in agricultural fields (Alvarado and Lopez, 1982; Hidalgo and Lopez, 1980a; Lopez and Azofeifa, 1981), and its dissemination does not seem to be associated with any particular set of environmental conditions.

Differences among three populations of M. javanica in total length, base of stylet to head end, DEGO, tail length, maximum body width, anal width and a ratio of second-stage juveniles, stylet, maximum body width, DEGO and spicules (measured as the chord of their arch as described by Chitwood (1949)) of males, and stylet and DEGO of females were reported also (Salazar, 1980a).

The differential host test (Taylor and Sasser, 1978) was performed also on a few populations of root-knot nematodes (Salazar, 1980a; Salazar and Lopez, 1980). In general, the reaction of the hosts could be considered "typical" for each of the species tested, except in the case of M. javanica, where one population was able to infect 'California Wonder' pepper (Salazar, 1980a), and the failure of several populations of M. hapla to reproduce on 'Tioga' strawberry (Salazar and Lopez, 1980). Race 1 of M. incognita was the only one found by Salazar and Lopez (1980).

The spatial distribution of Meloidogyne spp. under field conditions is another aspect that was studied in some detail. The densities of









M. incognita (Gonzalez, 1978a) and of a mixture of M. incognita and M. hapla (Perlaza et al., 1978; Perlaza et al., 1979) in vegetable fields varied greatly even in adjacent small plots. Similar observations were performed on rice regarding an undescribed species of Meloidogyne (Lopez, 1981a). This nematode was also concentrated in the upper 15 cm soil layer, and its density decreased sharply as sampling depth increased.

In burley tobacco, densities of second-stage juveniles of M.

incognita were greater in the horizontal plane 5-10 cm away from the trunk, and in the upper 15 cm soil layer. Densities decreased as sampling depth increased, but not to a large degree. These data were taken one week after harvest (Lopez, 1981b).

In sugarcane, higher densities of Meloidogyne spp. were located 15 cm away from the plants in the horizontal plane; vertically, the highest densities were located between 61 and 75 cm deep (Salazar, 1980b).

The influence of soil type and extraction method on the recovery of Meloidogyne spp. was studied. Significantly higher densities of M. incognita juveniles were extracted from an Ustropept soil when it was washed in water three times. Higher numbers were recovered from Ustult and Distropept soils when they were suspended 20 and 60 seconds in water, respectively, before being poured through the sieves. An arrangement of one 50-mesh sieve nested on top of two 325-mesh sieves and a

1.12 sp. g. sugar solution recovered higher densities from the Ustropept and the Distropept soils, respectively. Significantly more juveniles were extracted from the three soil types with the centrifugal-flotation technique than with the modified Baermann funnel (Alvarado and Lopez, 1982).









Different variations of both the centrifugal-flotation and the

modified Baermann funnel did not improve the extraction of juveniles of an undescribed species of root-knot nematode from a rice field. More juveniles were recovered with the centrifugal-flotation method (Alvarado and Lopez, 1981).

Another interesting observation on root-knot nematodes was that in four females of M. incognita some eggs developed to second-stage juveniles while they remained in the uterus (Perlaza and Lopez, 1979).

Materials and Methods

Nematode Populations

Sixteen populations of root-knot nematodes were collected from different localities in Costa Rica (Fig. 1) and increased in a greenhouse at the Facultad de Agronomia, Universidad de Costa Rica, San Pedro. Some selected ecological characteristics of the collection sites of these populations, along with the hosts on which they were collected and their population designation, are presented in Table 1.

The inoculum for the propagation of each population consisted of

several dozen egg masses collected from roots of the host from the original locality where the population was collected. Most populations were increased on tomato, cv. Rutgers. Populations CR7 and 9 were maintained on coffee, cv. Caturra, whereas population CR2 was maintained on rice, cv. C.R. 1113. All plants were grown in 2,000 cm3 clay pots that contained 1,700 cm3 of an Andept soil (43.2:31.4:25.4% sand:silt:clay; 8.7% 0.M. and 5.8 pH). The soil in all cases was treated with steam at 105*C for 24 hours prior to use. Each pot was fertilized twice a week during the first five weeks of plant growth with 150 ml of a 1% 20-20-20 fertilizer formula solution. Air temperatures varied between 17 and 310C.








12































84* 00'
Nicaragua



L-,- Caribbean Sea

01e




514
10' 0a t2 5o, 10



03



Pacific a
n
Ocean *im 17 r a
*

21
COSTA RICA
00
KILOMETERS

84*00





Fig. 1. Map of Costa Rica showing the approximate

location of the collection sites of 16
populations of Meloidogyne spp..











Table 1. Designations, sources and selected ecological characteristics of the collection sites of 16
populations of Meloidogyne spp. from Costa Rica.


Soil (%) Soil Elevation
Pop. Host Sand Silt Clay 0.M. pH m.a.s.l. Locality Ecological zone


CR1 Lycopersicon
esculentum Mill. 50.2 29.7 20.1 8.8 6.1 810 La Guacima Premontane moist forest CR2 Oryza sativa L. 75.2 14.7 10.1 3.4 6.0 22 La Cuesta Premontane wet forest, basal belt
CR3 Nicotiana
tabacum L. 68.2 13.7 18.1 3.2 5.4 550 Repunta Tropical moist forest CR4 Carica papaya L. 55.2 26.7 18.1 2.4 6.2 42 Ciudad Neilly Tropical wet forest CR5 Carica papaya L. 30.1 31.8 38.1 5.6 6.0 220 Orotina Tropical moist forest CR6 Lycopersicon
esculentum Mill 35.1 46.8 18.1 2.7 6.1 905 Santa Ana Premontane moist forest CR7 Coffea arabica L. 44.8 32.7 23.5 8.7 6.1 1,360 San Luis Premontane moist forest CR9 Coffea arabica L. 32.1 34.8 33.1 11.6 6.2 1,020 Sarchi Premontane wet forest CR10 Eupatorium
subcordatum 88.1 5.8 6.1 2.8 6.2 2,400 Division Montane rain forest CR11 Musa acuminata X
M. balbisiana,AAB 52.1 12.7 35.2 4.3 6.0 60 Palmar Norte Premontane wet forest CR12 Carica papaya L. 26.1 31.8 42.1 5.3 6.1 10 Paquera Premontane moist forest CR13 Bidens pilosa L. 35.5 58.9 5.6 9.2 6.0 1,940 Porrosati Lower montane rain forest CR14 Brassica oleracea
var. capitata L. 29.0 43.0 28.0 8.4 6.0 2,050 El Empalme Lower montane wet forest CR15 Impatiens
balsamina L. 34.0 33.0 33.0 6.8 6.2 2,200 Palmira Lower montane wet forest CR16 Musa acuminata X
M. balbisiana,AAB 80.6 8.0 11.4 5.6 5.8 75 Rio Frio Tropical wet forest CR17 Carludovica sp. 55.1 25.8 19.1 8.6 6.1 10 Piedras Blancas Tropical wet forest









Morphology

Twenty specimens were used for each character studied in the males, females and infective second-stage juveniles. All measurements were analyzed statistically with a one-way classification model, and the mean values were compared using the Duncan's Multiple Range Test.

The perineal patterns were prepared according to the method

described by Franklin (1962) and later modified by Taylor and Netscher (1974), but without staining the roots. Each perineal pattern was divided into several zones, following the method described by Esser et al. (1976).

To obtain infective second-stage juveniles, eggs from several egg masses collected at random from the host roots were kept in a small petri dish for 24 hours. Live infective second-stage juveniles were placed on glass slides in a drop of distilled water, ringed with Zut and a coverslip applied. Fifteen to 20 minutes after mounting, the juveniles were observed with an ordinary compound microscope (LM). Juveniles lying in a straight plane were measured and data recorded.

Males were obtained by dissecting galled roots in tap water. The males were mounted using the same method as described for the juveniles. Occasionally, the slide was maintained for 24 hours to facilitate the observation of the lateral fields.

All measurements were made with a calibrated ocular micrometer at 1,500X magnification (oil immersion objective), except for the female length and maximum body width, and the second-stage juvenile length, which were measured at 150X.









Differential Plants

The reactions of seven differential plants (Taylor and Sasser, 1978) were evaluated for each of the 16 populations of root-knot nematodes. Plants used were tomato, cv. Rutgers; tobacco (Nicotiana tabacum L.), cv. NC-95; pepper (Capsicum annum L.), cv. California Wonder; corn (Zea mays L.), cv. Minnesota A-401; cotton (Gossypium hirsutum L.), cv. Deltapine 16; peanut (Arachis hypogea L.), cv. Florunner, and watermelon (Citrullus vulgaris Schard), cv. Charleston Grey.

Seeds of the plants, except tobacco, were germinated over filter paper in petri dishes at 28*C for three days; the germinated seeds were then placed in small plastic pots containing steam-pasteurized soil. Seedling ages at inoculation were as follows: corn--10 days old, peanut, cotton and watermelon--20 days old, tomato--30 days old, and tobacco and pepper--60 days old. Inocula of all populations, except CR2, 7 and 9, were obtained from a galled root system of tomato, which was cut into small pieces and treated with a 1% sodium hypochlorite solution (Hussey and Barker, 1973). Coffee roots infected with populations CR7 and 9 were cut into small pieces and then chopped in a blender for 20 seconds; the resulting material was treated with the 1% sodium hypochlorite solution. The galled rice roots used to collect the inoculum of CR2 were macerated in a blender for 15 seconds and then washed with tap water on a 200-mesh sieve nested on top a a 500-mesh sieve; a strong jet of water was applied and the eggs were recovered from the 500-mesh sieve.

About 10,000 eggs were pipetted over 1,000 cm3 of soil, the seedlings transplanted and 700 cm3 of soil added. Pots were placed randomly on the greenhouse bench, and each population was isolated by plastic









dividers to avoid contamination. Each host-population combination was replicated four times.

Fifty-five to 60 days after inoculation the plant roots were washed free of soil and immersed for 30 minutes in a 0.0016% Phloxine B solution (Dickson and Struble, 1965) to stain the egg masses. Each root system was rated according to the number of egg masses, using the following scale (Taylor and Sasser, 1978):

0: 0 egg masses 3: 11-30 egg masses 1: 1-2 egg masses 4: 31-100 egg masses

2: 3-10 egg masses 5: more than 100 egg masses.

In certain cases, perineal patterns were prepared from females on

certain differential plants to insure that the species recovered was the same used for the inoculation.

Results

Five species of Meloidogyne were identified among the 16 populations studied. These species were M. incognita (populations CR1, 3, 5, 6, 11, 12, 16, and 17), M. exigua (populations CR7 and 9), M. hapla (populations CR10, 14, and 15), M. arenaria (population CR4), and an undescribed species (population CR2). Population CR13 was identified as a mixture of M. incognita and M. hapla. Morphology

The interpretation of the predominant type of perineal pattern for each species is presented in Table 2. Only specimens of the undescribed species (CR2) and a few from M. exigua (CR7 and 9) had a posterior protuberance. Populations CR16 and 17 of M. incognita had a few striae originating at the vulval lips and going out to the sides. M. exigua (CR7 and 9) had three striae in the perineum, whereas M. hapla (CR10,










Table 2. Interpretation of the predominant type of perineal pattern of
females of 16 populations of Meloidogyne spp. from Costa Rica.



Vulva
Posterior lip Perineum Lateral Striae zone Pop. protuberance striae striae incisures 1 2 3 4


M. incognita
CR1 CR3 CR5 CR6 CR11 CR12 CR16 CR17

M. exigua
CR7 CR9

M. hapla
CR10 CR14 CR15

M. arenaria
CR4

M. sp. CR2

M. incognita
& M. hapla CR13


F FWB F FWB F FWB F FWB F FWB F MWB M MWB F FWB


FWB FWB FWB FWB FWB MWB MWB
FWB


FWB
FWB FWB
FWB FWB MWB MWB
FWB


F FSB FSB FSB F FSB FSB FSB


F FSU F FSU F FSU


A



1 &A


FSU FSU FSU FSU FSU FSU


F FSB FSB FSB F FSU FSU FSU


I &A


F FSW& FSU& FSU&
FWB FWB FWB


A: absent; P: present; F: few; M: moderate in number; W: wavy; B: broken; U: unbroken; S: smooth; I: interrupted.









14, and 15) and some specimens of CR13, a population consisting of a mixture of M. incognita and M. hapla, had one stria. The striae of M. arenaria (CR4), M. exigua (CR7, 9) and M. hapla (CR10, 14, 15) were interrupted where the lateral lines normally are, but they were not distinct enough to be considered lateral lines. The M. incognita populations CR1, 3, 5, 6, 11, and 17 had a few, wavy and broken striae in zones 2, 3, and 4, whereas CR12 and 16 had a moderate number of striae in these zones. In these same zones M. exigua (CR7, 9) had few, smooth, broken striae, whereas M. hapla (CR10, 14, 15) had few, smooth, unbroken striae. M. arenaria (CR4) had few, smooth, broken striae in zones 2, 3, and 4. In the mixture of M. incognita and M. hapla (CR13) perineal patterns with few, smooth and unbroken striae, as well as perineals with few, wavy, broken striae in zones 2, 3, and 4 were found. The undescribed species (CR2) had few, smooth, mostly unbroken striae in zones 2, 3, and 4. Striae of the undescribed species and of M. hapla were relatively fine whereas they were relatively coarse in the other species.

The shape of the perineal pattern varied with the species. In M. incognita the perineal patterns of all populations were mostly pyriform, with a trapezoidal dorsal arch. The perineal patterns of the two M. exigua populations were roughly rounded, with a low rounded dorsal arch; the striae were rather coarse and far apart. In M. hapla populations the perineal patterns were roughly rounded, with a low and wide dorsal arch. No wings were observed in the perineal patterns, but punctations on the tail terminus area were present. The striae were closely spaced. In M. arenaria the perineal patterns were mostly oval, with striae forming a shoulder on the low, flat to rounded dorsal arch. In the mixture










of M. incognita and M. hapla both pyriform and roughly rounded perineal patterns were found. The undescribed species (CR2) had oval shaped perineals, with high and wide rectangular dorsal arches; the striae were far apart. A photomicrograph of one perineal pattern from each population, except of the mixture of M. incognita and M. hapla, is presented in Figs. 2 and 3.

The mean values of morphometric characters of the females are

presented in Table 3. Highly significant differences among populations were found in stylet, DEGO, distance between the middle of the excretory pore and the head end (excretory pore), maximum body width, body length, vulva, anus-vulva and interphasmidial distances. The ranges for these characters are presented in Table 15 in the appendix. In general, the ranges of all characters overlapped to some degree among populations.

Average values for the characters measured in infective secondstage juveniles are presented in Table 4, and their ranges are presented in Table 16 in the appendix. Highly significant differences among populations were found in total length, tail length, maximum body width, anal width, stylet base to head end, DEGO, and the a and c ratios. Undilated recta were present in M. exigua (CR7, 9) and M. hapla (CR10, 14, 15), whereas they were dilated in the other populations. In all juveniles the hemizonid was located anterior to the excretory pore. The range of total length of M. exigua (CR7, 9) did not overlap with that of the undescribed species (CR2). A similar situation was found between the M. hapla populations CR14 and 15 when compared to the undescribed species (CR2). Range values for the M. hapla population CR10 did overlap with that of the undescribed species, although it did not with the range values of the other two populations of M. hapla (CR14, 15). There





















































Fig. 2. Photomicrographs of female perineal patterns
of nine populations of Meloidogyne spp. from
Costa Rica. CR4: M. arenaria. CR1, 3, 5, 6, 11,
12, 16, and 17: M. incognita.

















































Fig. 3. Photomicrographs of female perineal patterns
of six populations of Meloidogyne spp. from Costa Rica. CR2: M. sp.. CR7, 9: M. exigua.
CR10, 14, and 15: M. hapla.










Table 3. Morphometric characters of females of 16
Meloidogyne spp. from Costa Rica.


populations of


S Body Body
Pop. Exc. pore Stylet DEGO width length


M. incognita **
CR1 23.7 a 15.2 cde 4.1 bc 486 efg 634 bcd CR3 26.4 abcd 14.3 abcd 4.0 abc 538 g 764 gh CR5 26.2 abcd 15.9 e 3.8 ab 459 def 679 cdef CR6 23.4 a 15.6 de 4.0 abc 519 g 721 fg CR11 25.5 abc 13.5 ab 3.9 ab 495 efg 784 h CR12 26.4 bcd 15.4 cde 3.4 a 439 de 656 bcde CR16 31.7 cde 15.4 cde 4.6 cd 371 bc 642 bcde CR17 24.7 ab 15.2 cde 4.3 bcd 471 def 659 bcde

M. exigua
CR7 35.3 e 14.8 bcde 6.1 e 272 a 493 a CR9 32.6 bcde 14.4 bcd 4.8 e 325 ab 491 a

M. hapla
CR10 34.8 e 12.9 a 5.7 e 472 def 697 ef CR14 36.5 e 13.7 ab 5.7 e 423 cd 629 bc CR15 33.3 de 14.2 abc 5.8 e 486 efg. 728 fg

M. arenaria
CR4 34.2 e 15.5 cde 4.6 d 508 fg 698 ef

M. sp.
CR2 35.4 e 13.6 ab 3.9 ab 468 def 602 b

M. incognita
& M. hapla
CR13 25.2 abc 15.6 de 4.2 bc 495 efg 690 def

CV (%) 28.4 10.1 14.6 14.6 9.9


DEGO refers
DEGO refers


to the distance between the base of the stylet knobs and


the dorsal esophageal gland orifice.


Mean of 20 observations. All measurements in Um. Means in the same column followed by the same letter do not differ significantly from one another according to Duncan's Multiple Range Test (P = 0.01).










Table 3-continued.


Interphasmidial
Pop. Vulva length Anus-vulva distance


M. incognita
CR1 CR3 CR5 CR6 CR11 CR12 CR16 CR17

M. exigua
CR7 CR9

M. hapla
CR10 CR14 CR15

M. arenaria
CR4

M. sp. CR2


M. incognita & M. hapla CR13


22.1 25.3
25.0 24.5 23.2
20.6 23.5 23.3


bcde
f
f
ef bcdef abcd def cdef


18.9 a 20.6 abcd


20.7
20.8 20.4


abcd
abcd abc


21.5 abcd 23.2 bcde


20.3 ab


17.8
18.9
18.6 17.0 19.8
17.5 18.1 18.0


cde ef def abcde
f
abcde cdef cde


17.2 abcde 16.7 abcd


15.7
15.6 17.6


ab
a bcde


17.8 cde 16.3 abc 17.5 abcde


CV (%) 14.2 11.7 7.9


*
DEGO refers to the distance between the
the dorsal esophageal gland orifice.


base of the stylet knobs and


Mean of 20 observations. All measurements in Um. Means in the same column followed by the same letter do not differ significantly from one another according to Duncan's Multiple Range Test (P = 0.01).


24.5
28.6 27.2 27.5 26.2
23.0 26.6 26.9


24.8 ef 22.6 bc 21.7 b 24.9 ef 22.8 bcd 27.6 gh 15.1 a 23.9 cde










Table 4. Morphometric characters of second-stage juveniles of 16
populations of Meloidogyne spp. from Costa Rica.



Total Tail Maximum Pop. length length body width Anal width


M. incognita
CR1 CR3 CR5 CR6 CR11 CR12 CR16 CR17

M. exigua
CR7 CR9

M. hapla
CR10 CR14 CR15

M. arenaria
CR4

M. sp.
CR2


M. incognita & M. hapla CR13


414 402 426 449 420 419 461 386


**
cd
c
d
e
d
d
e
b


373 ab 368 a 464 f 373 ab 373 ab 459 e 466 f 418 d


53.3
49.8 53.9
57.5 52.8
49.8 56.4 47.4


48.5 a 48.8 a


61.2 47.4
48.1


57.3 d 69.5 f


14.9
16.1 15.2 14.9 15.1 15.6 14.9
15.7


ab c
e
bcd abc bcd cde abc de


14.5 ab 14.7 ab


14.7 14.6
14.5


14.2 a 16.1 e


54.7 bcd 14.9 abc


CV (%) 4.0 6.3 5.5 12.0


*
DEGO refers to the distance between the base the dorsal esophageal gland orifice.


of the stylet knobs and


Mean of 20 observations. All measurements in Um. Means in the same column followed by the same letter do not differ significantly from one another according to Duncan's Multiple Range Test (P = 0.01). All juveniles had the hemizonid anterior to the excretory pore. In CR7, 9, 10, 14, and 15 the recta were undilated, whereas in the remaining populations they were dilated.


10.8 11.1 10.8 10.9 11.1 10.3 9.0
10.3


cde de cde cde de
bcde
a
bcde


9.4 ab 9.4 ab


10.6 bcde 10.2 bcd 10.0 abcd


9.8 abc 11.4 e


10.3 bcde









Table 4-continued.


Stylet base
Pop. to head end DEGO a c


M. incognita
CR1 15.3 de 3.2 ab 27.9 bcd 7.8 bcde CR3 15.6 ef 3.1 ab 26.2 abc 8.1 ef CR5 15.7 ef 3.0 a 28.3 bcd 7.9 cdef CR6 15.5 ef 3.1 ab 30.3 def 7.8 bcde CR11 15.1 cd 3.1 ab 28.0 bcd 8.0 def CR12 15.4 def 3.8 d 23.5 a 8.4 g CR16 15.8 f 3.6 cd 31.2 def 8.2 fg CR17 15.5 ef 3.2 ab 25.0 ab 8.2 fg

M. exigua
CR7 13.6 a 3.2 ab 25.6 ab 7.7 bcd CR9 13.6 a 3.5 bcd 25.2 ab 7.5 b

M. hapla
CR10 14.8 c 4.5 e 31.7 ef 7.6 bc CR14 14.2 b 4.2 e 25.7 ab 7.9 cdef CR15 14.0 b 3.7 d 25.8 ab 7.8 bcde

M. arenaria
CR4 15.4 def 3.5 bcd 32.6 f 8.0 def

M. sp.
CR2 14.2 b 3.3 abc 29.2 cde 6.7 a

M. incognita & M. hapla
CR13 15.3 de 3.1 ab 28.2 bcd 7.6 bc

CV (%) 3.2 12.7 13.5 4.7


DEGO refers to the distance the dorsal esophageal gland


between the base of orifice.


the stylet knobs and


**
Mean of 20 observations. All measurements in Um. Means in the same column followed by the same letter do not differ significantly from one another according to Duncan's Multiple Range Test (P = 0.01). All juveniles had the hemizonid anterior to the excretory pore. In CR7, 9, 10, 14, and 15 the recta were undilated, whereas in the remaining populations they were dilated.










was no overlap in the range of total length either between M. arenaria (CR4) and M. exigua (CR7, 9), or between M. arenaria (CR4) and the M. hapla population CR15.

Tail length ranges did not overlap when the undescribed species was compared to M. exigua (CR7, 9), two M. hapla populations (CR14, 15) and five M. incognita populations (CR1, 3, 11, 12 and 17). The remaining range values of all characters overlapped among populations of the different species studied.

Average values and observations of certain male characters are presented in Table 5. The ranges are presented in Table 17 in the appendix. All males had areolated lateral fields, although to a variable degree. In most populations they had four lines in the lateral fields, although five were also observed in some specimens of CR14, a population of M. hapla. Only one gonad was observed in males of the undescribed species (CR2), the M. incognita populations CR3 and 11, and the M. hapla population CR15; the others had a varying percentage of males with two gonads.

Highly significant differences were found among populations in the stylet, DEGO and spicules (chord of arch). The mean stylet length of the undescribed species was the lowest, followed only by those of M. exigua (CR7, 9), which had stylets 2.4 and 2.9 um longer, respectively. The stylet range of the undescribed species (CR2) overlapped with those of M. exigua (CR7, 9), while these two did not overlap with the range values of the M. incognita populations CR1, 3, 5, 6, 16 or 17. The ranges for the DEGO and spicule measurements overlapped among the different populations.





27



Table 5. Morphological characters of males of 16 populations of
Meloidogyne spp. from Costa Rica.



Spicules Number of % males (chord of lateral with ye Pop. Stylet arch) DEGO Areolation lines gonad


M. incognita CR1 23.6 CR3 25.1 CR5 24.1 CR6 25.0 CR11 22.0 CR12 22.6 CR16 24.4 CR17 25.7


f hi fg ghi
e
e fgh
i


18.4 b 18.9 b


21.7 20.4 20.5


M. arenaria
CR4 24.3 fgh


16.0 a


35.0
34.5 32.6 34.2
33.5 31.1 32.3 35.4


hi ghi efg
fghi fghi de ef
i


24.1 a 25.0 a


30.0 29.0
27.7


33.0 efgh


27.2 b


3.5b
3.0
2.8 2.9
2.8 3.0
3.2 3.5


yes yes yes yes yes yes yes yes


3.2 ab yes 4.9 de yes


5.0 e 4.1 c 5.0 e


95 100
65 85 100
80 70 95


4
4


4 4-5
4


3.1 ab yes 3.3 ab yes


M. incognita
& M. hapla
CR13 21.0 cd


28.2 bc 4.4 cd yes


CV (%) 6.8 9.3 16.9


DEGO refers to the distance between the base of the stylet knobs and
the dorsal esophageal gland orifice.

The rest of the males had two gonads.

Mean of 20 observations. All measurements in Pm. Means in the same
column followed by the same letter do not differ significantly from
one another according to Duncan's Multiple Range Test (P = 0.01).


M. exigua CR7 CR9

M. hapla CR10
CR14 CR15


M. sp.
CR2









Differential Plants

Similar responses were obtained in the four replicates of each differential plant-population combination, and the average values are presented in Table 6. The differential plant responses indicated that populations CR1, 5, 6, and 17 were M. incognita race 1, populations CR3, 11, 12, and 16 were M. incognita race 2, population CR4 was M. arenaria race 2, populations CR7 and 9 were M. exigua, populations CR10, 14, and 15 were M. hagla, population CR2 was a different species, and population CR13 was a mixture of M. incognita and M. hapla.

The responses to the differential plants gave evidence of pathogenic variation in M. exigua. The two populations of this species could be differentiated by their ability or inability to infect tomato, cv. Rutgers (Table 6). Population CR9 was able to reproduce well on this host but CR7 was not.

A major difference among populations of M. hapla was found in the reaction of pepper, cv. California Wonder. Populations CR14 and 15 reproduced abundantly on this host, but CR10 reproduced only to a limited extent.

Tomato was heavily infected and received the maximum rating value of 5 with all but the undescribed species (CR2) and the M. exigua population CR7. Tobacco was not infected by the undescribed species (CR2) and M. exigua (CR7, 9), only slightly by the M. incognita populations CR5, 6, and 17, and heavily by the remaining populations. Pepper was not a host for the undescribed species (CR2), and was infected only slightly by CR10, a population of M. hapla; the other populations reproduced well on this host. Cotton was not infected, except slightly by CR6 and 16, two populations of M. incognita. Peanut was a good host for










Table 6. Response of seven differential plants to 16 populations of Meloidogyne spp. from Costa Rica.


Pepper Cotton Watermelon Corn Tomato Tobacco 'Califorinia 'Delta- Peanut 'Charleston 'Minnesota Pop. 'Rutgers' 'NC-95' Wonder' pine 16' 'Florunner' Grey' A-401'

M. incognita
CR1 5 3 5 0 0 5 5 CR3 5 5 5 0 0 4.5 5 CR5 5 1.5 4.5 0 0 3 3.2 CR6 5 1.5 5 1.5 0.2 5 4.7 CR11 5 5 5 0 0 5 5 CR12 5 4 3.5 0 0 5 1.5 CR16 5 5 5 0.5 0 5 5 CR17 5 2 5 0 0 5 3.7 M. exigua
CR7 1.5 0 5 0 0 4 0 CR9 5 0 4.7 0 0 4 0 M. hapla
CR10 5 5 1.7 0 5 5 0.2 CR14 5 4 5 0 5 0 0 CR15 5 5 5 0 5 0 1.6 M. arenaria
CR4 5 5 5 0 0 5 3.2 M. sp. **
CR2 0 0 0 0 0 0 0 M. incognita
& M. hapla
CR13 5 4.5 5 0 2.7 2 2.5
Mean of four replicates. Responses evaluated according to the number of egg masses/root system.
**0 = 0 egg masses; 1 = 1-2; 2 = 3-10; 3 = 11-30; 4 = 31-100; and 5 = more than 100 egg masses. Inoculum viability was evidenced by high reproduction on rice plants inoculated at the same time as
the differential plants.









M. hapla (CR10, 14, 15), moderate for the mixture of M. incognita and M. hapla (CR13) and a poor host for CR6, a population of M. incognita. Watermelon was not infected by the undescribed species (CR2), two populations of M. hapla (CR14, 15), and only lightly by the third population of M. hapla (CR10) and by the mixture of M. incognita and M. hapla. This plant was a good host for the other populations. Finally, corn was not a host for the undescribed species (CR2), M. exigua (CR7, 9), and for two populations of M. hapla (CR10, 14), a poor host for M. arenaria (CR4), the mixture of M. incognita and M. hapla (CR13), one population of M. hapla (CR15) and two of M. incognita (CR5, 17), and a good one for the other populations.

Discussion

Morphology

M. incognita.

The general shape of the perineal patterns was similar among the populations studied and could be used to distinguish this species from the others. The interpretation of the characteristics exhibited by the perineal patterns agreed with the reports by previous authors in Costa Rica (Hidalgo and Lopez, 1980a; Salazar and Lopez, 1980). Similarly, the absence of a posterior protuberance was noted. It was noticed that when the mean values of the morphometric characters of each population were compared to those previously reported from Costa Rica, the juvenile length and the female stylet in CR1 were greater. In CR3 the juveniles were wider; CR5 had juveniles with a greater length and females with a longer stylet; CR6 had longer juveniles, with longer tails and a greater a ratio; population CR11 had longer juveniles, and the interphasmidial distance, tail length, stylet base to head end, DEGO and the a ratio in









the females were slightly greater. They also had a smaller anal width. In the females, the excretory pore was longer.

Males of all populations had mean values of their characteristics similar to those reported by these authors (Hidalgo and Lopez, 1980a; Salazar and Lopez, 1980).

M. exigua.

There are no previous reports about the morphometrics of this

species in Costa Rica, so comparisons were made to the data provided by Chitwood (1949), Lordello and Zamith (1958), Esser et al. (1976), and Whitehead (1968). The general shape and the characteristics of the striae of the perineal pattern agreed with previous descriptions. A few females of each Costa Rican population had the neck region located on the ventral side of the body and a posterior protuberance. This observation contradicts the statement of Esser et al. (1976) and that of Whitehead (1968), who denied the presence of such protuberance on females of this species.

On the other hand, some males of the two populations from Costa

Rica had twisted bodies and some untwisted bodies. This observation was in agreement with the previous report by Scotto la Massese (1969), and contradicts the statement by Lordello and Zamith (1958), that males of M. exigua did not have a twisted body, thus constituting an exception among the root-knot nematodes. Another contradiction with the report by Lordello and Zamith (1958) was the finding of only one testis in some males of both populations. Lordello and Zamith (1958) reported that all males possessed two testes.

The two Costa Rican populations had longer infective second-stage juveniles and females with longer stylets than previously reported.









Males of CR7 had greater DEGO values than those reported by Lordello and Zamith (1958). All other values found in this investigation agreed with, and in some cases were identical to, those previously reported.

M. hapla.

The finding of this species outside the Central Plateau and the

Central Volcanic Range, the only areas where it had been found previously (Lopez and Salazar, 1978; Mattey and Lopez, 1978; Lopez and Azofeifa, 1981; Salazar and Lopez, 1980) widens its reported geographical distribution in Costa Rica. Both El Empalme and Division are high altitude areas with high precipitation and relatively cool temperatures all year round. This agrees with the observed tendency for the distribution of M. hapla in the rest of Costa Rica (Lopez and Salazar, 1978).

The shape of the perineal patterns and the characteristics of their striae were in close agreement with previous reports from Costa Rica (Lopez and Salazar, 1978; Salazar and Lopez, 1980), except that no wings were observed in the perineal patterns. Females of the three populations had greater values for the excretory pore and the DEGO than those reported for other Costa Rican populations (Lopez and Salazar, 1978; Salazar and Lopez, 1980). The population CR10 had greater values for the total length and tail length of the juveniles, and for the stylet and spicules (chord of arch) of the males. The CR14 population had longer spicules than found by previous authors. The other characters had mean and range values similar to those reported earlier (Lopez and Salazar, 1978; Salazar and Lopez, 1980).

The recta of all juveniles were undilated. When first found in Costa Rica, Lopez and Salazar (1978) observed some juveniles with dilated recta in a population collected from cabbage. Later, these









authors (Salazar and Lopez, 1980) found M. incognita and M. hapla coexisting in cabbage in the same general area of their first finding. Since M. incognita juveniles have dilated recta (Chitwood, 1949), the possibility of a mixture of both species in the first report seems likely, and therefore makes the report of dilated recta in M. hapla juveniles doubtful.

M. arenaria.

The finding of a population of M. arenaria in Ciudad Neilly is the first report of this species in Costa Rica. Comparisons were made to the values and observations given by previous authors (Chitwood, 1949; Esser et al., 1976; Eisenback et al., 1981). The general shape and characteristics of the striae of the perineal pattern were similar to those reported by these authors, but the second-stage juveniles were shorter than the value given by Eisenback et al. (1981), although similar to the values given by Chitwood (1949). All other morphometric values for females, males and juveniles were similar to the reports by the previously mentioned authors.

Meloidogyne sp. n.

The females of this root-knot nematode could be differentiated from the other species by the presence of a posterior protuberance and the neck and head regions located on the ventral side of the body. The body was usually oval, in contrast to the pyriform shape found in M. exigua, some of which showed a posterior protuberance and the neck on the ventral side of the body. Some, but not all specimens of M. exigua exhibited these same characters.

The shape of the perineal pattern was also unique. Other differentiating characters were the short interphasmidial distance in the









females, the longer juvenile tails, the smaller c ratio of juveniles and the shorter male stylet. The phasmids of the females were also smaller than in other root-knot nematode species. Differential Plants

As pointed out by Sasser and Carter (1982), differential plants 1) provide a preliminary or corroborative indication of the root-knot nematode species being evaluated, based on the usual response of the hosts, and 2) detect pathogenic variation of a population, as determined by host responses different from the usual for the various species. However, differential plants cannot be relied upon entirely for identification, because the population being studied may be a mixture of species or a species for which no or limited host response data are available.

For example, the undescribed species from rice did not reproduce on any of the differential plants (Table 6). The reaction of the plants, however, was used to differentiate among the other species studied, and even for the determination of the host race among populations of M. incognita and M. arenaria.

Based on the scheme provided by Sasser and Carter (1982), the M. incognita populations CR1, 5, 6, and 17 were designated as race 1, whereas populations CR3, 11, 12, and 16 were designated as race 2. This is the first report of the presence of race 2 in Costa Rica. Salazar and Lopez (1980) had previously reported race 1 only.

In spite of the evidence of pathogenic variation in the two populations of M. exigua, it seems premature at this time to call them races. This term was applied to populations of Meloidogyne species that were shown by numerous experiments to have unique host preferences, and that









were named only after there was evidence of wide geographical distribution and/or sufficient significance for crop rotation and/or plant breeding programs (Taylor and Sasser, 1978; Sasser and Carter, 1982). Most of the criteria used for the application of the term host race were not fulfilled in this case. Future work could give the necessary proof that they indeed deserve to be designated as host races. From a practical point of view, this finding could be of value to farmers in the area of Sarchi, as some fields where coffee was grown were changed to tomato production.

Differences in the ability of M. hapla populations to reproduce on pepper were found. Reactions of the other differential plants to the three populations were similar, and agreed with the usual response given by them to this species (Taylor and Sasser, 1978; Sasser and Carter, 1982). As in the case of M. exigua, it seems premature at this time to apply the term host races to these populations.

The population of M. arenaria, similar to most of the populations in the world collection of the International Meloidogyne Project (Sasser and Carter, 1982), did not reproduce on peanut, cv. Florunner, and therefore was determined to be race 2 of this species.

The reaction given by the differential plants to CR 13, the mixture of M. incognita and M. hapla, was different from the usual one given to each of the major species (Sasser and Carter, 1982). Previous workers have found this same mixture of species in the Central Volcanic Range of Costa Rica, on plants such as cabbage, carrot, lettuce and green peas (Lopez and Azofeifa, 1981; Padilla and Lopez, 1979; Perlaza et al., 1978; Perlaza et al., 1979; Salazar and Lopez, 1980). As pointed out by several of them, such mixture of species makes the management of







36


root-knot nematodes by crop rotation and resistant cultivars even more difficult, and might require some long term studies for the development of profitable management schemes.
















CHAPTER III
DESCRIPTION OF Meloidogyne salasi sp. n.

Introduction

In 1968, a root-knot nematode causing severe damage on upland rice (Oryza sativa L.) was found in Volcan de Buenos Aires, Puntarenas, Costa Rica. The parasite was tentatively identified as a new species of Hypsoperine (Figueroa, 1973). Although several aspects of the biology, morphology and the pathogenicity of this nematode on rice were studied, no species description was given. In late 1979, high population densities of an undescribed root-knot nematode were found on rice, cv. C.R.1113, at La Cuesta, Puntarenas, Costa Rica (Alvarado and Lopez, 1981). This nematode caused severe damage on rice growing in the field and also was highly pathogenic to rice grown under greenhouse conditions. The nematode was localized on a few farms in the southeastern part of the country (Sancho, 1981). In addition, a root-knot nematode was reported infecting rice in the province of Codcle, Panama (Tarte, 1981). Because of the severe damage caused by the nematode, farmers in the region abandoned rice production in favor of grasslands. Again, no description of the species was given (Tarte, 1981). An examination of a few perineal patterns from the population studied by Figueroa (1973) and some preserved specimens from Panama provided by Ing. Julio Lara, confirmed that the species involved was the same as the root-knot nematode found on rice at La Cuesta, Costa Rica.









Populations of this root-knot nematode from both Costa Rica and Panama were studied cytologically (Triantaphyllou, 1982); both populations reproduce by obligatory mitotic parthenogenesis and both have a diploid chromosome number of 36.

The nematode is described, illustrated and named herein as Meloidogyne salasi sp. n., in honor of Professor Luis Angel Salas Fonseca, the founder of Plant Nematology in Costa Rica.

Materials and Methods

A culture of M. salasi sp. n., increased and maintained on rice, cv. C.R.1113, was established from eggs and infective second-stage juveniles obtained from the type locality of La Cuesta, Costa Rica. Nematodes from this culture were used for all morphological studies. Light Microscope (LM) Studies

Galled rice roots were cut open in shallow petri dishes containing distilled water. Eggs were placed on a glass slide, ringed with Zut and covered with a coverslip. Other eggs were left overnight in the petri dish and the freshly hatched second-stage juveniles were placed ten per slide in a drop of water contained in a ring of Zut and a coverslip applied. Approximately 20 minutes after mounting the juveniles were observed and measured using a camera lucida.

Males were dissected from old galls and prepared using the same method as described for the juveniles.

Females were dissected from galled roots boiled in lactophenol for two minutes. The perineal patterns were prepared according to the method described by Franklin (1962) and modified by Taylor and Netscher (1974). Whole females were mounted on a cavity slide and their length (excluding neck) and maximum body width were drawn. The females were removed from









the solution and punctured near mid-body with a fine needle to release the internal pressure. The head and the neck were excised and mounted in a drop of lactophenol on a slide, a coverslip added and ringed with Zut . Type specimens of males and females were fixed in 3% formalin for 48 hours, transferred to lactophenol at 50*C for 24 hours, and mounted in dehydrated glycerin.

Nomarski differential interference optics was used to observe all specimens. Photomicrographs of males, second-stage juveniles and the perineal patterns of females were taken with an Olympus OM-2 camera. Drawings of males, females and second-stage juveniles were prepared with a camera lucida.

Scanning Electron Microscopy (SEM) Studies

Males, females and second-stage juveniles were processed for SEM by a modification of the techniques described by Eisenback and Hirschmann (1979, 1980), and Eisenback et al. (1980).

Freshly hatched second-stage juveniles were obtained from eggs placed in distilled water for 18-24 hours at room temperature. The juveniles were transferred to 0.5 ml of distilled water, chilled at 5*C for one hour, and killed by adding three drops of cold (50C) 4% glutaraldehyde solution buffered with 0.1 M sodium cacodylate at pH 7.1. More buffered 4% glutaraldehyde was added at 24-hour intervals, three drops at a time, until a final 2% concentration was obtained. Fixation continued for an additional 72 hours at 50C. The juveniles were washed two times in sodium-cacodylate buffer (pH 7.1), transferred to a small plastic chamber with a fine screen (15 um diameter pore) on the bottom and kept for 24 hours at 50C. Postfixation was done with 2% osmium tetroxide, buffered with 0.1 M sodium cacodylate at pH 7.1, for 18-24









hours at room temperature. This solution was replaced with sodiumcacodylate buffer and kept at 50C for another 24 hours. Specimens were dehydrated with a graded series of ethanol (5-20-20-35-50-65-95-100%) at room temperature, with 24-hour intervals per step. Another screen was placed on top of the chamber, and the entire contents critical-point dried with CO2 in a Balzer drier. Dried nematodes were position with one third of their body (the anterior or the posterior part) lying across a human hair that was placed on the surface of a stub covered with double-coated tape. They were coated with gold for five minutes in a Giko Engineering 1 B-2 model ion coater and viewed with a Hitachi S-450 scanning electron microscope operated at 20 KV of accelerating voltage. Type 55 Polaroid film was used for photomicrographs.

Males were dissected from galled rice roots and treated as described previously for second-stage juveniles.

Small pieces of galled roots containing females of M. salasi sp. n. were fixed in a 4% glutaraldehyde solution buffered with 0.1 M sodiumcacodylate at pH 7.1. After 6-7 days whole females were dissected from the roots and prepared as described previously for juveniles.

In describing the external morphology of males, females, and secondstage juveniles, the terminology proposed by Eisenback and Hirschmann (1979, 1980) and Eisenback et al. (1980) was followed.

Species Description

Meloidogyne salasi sp. n.

Females. Measurements of 50 females in lactophenol are presented in Table 7.

Measurements of holotype in glycerin. Body length (excluding neck): 422 pm; maximum body width: 306 pm; neck length: 133 pm; neck width at









Table 7. Measurements of 50 females and eggs of Meloidogyne salasi
sp. n. from rice, cv. C.R.1113.



Standard Standard error of deviCharacter Mean Range the mean ation CV (%)


Female linear measurements (um)


Body length Maximum body width Neck length Neck width at middle of metacorpus Middle of metacorpus to head end
Metacorpus width Metacorpus length Metacorpus valve width Metacorpus valve length Stylet
Stylet knobs height Stylet knobs width DEGO
Excretory pore-head end Vulva slit length Anus-vulva Interphasmidial distance


Female ratios
a
Body length/neck length Stylet knobs width/height Metacorpus length/width Metacorpus valve length/width

Egg linear measurements (um) Length
Width


Egg ratios Length/width


486.3 338.1
135.1


372.0-625.0 209.0-425.0
86.0-203.0


63.3 43.7- 99.9


78.2 35.7 35.6
10.6 13.7 10.0 2.1 3.4 4.9
32.1 21.9 16.4 15.2


60.9
29.0
30.0
9.0
11.5
8.1
1.5
2.5
3.4
18.7
15.9
9.0
10.6


1.0
2.1
0.8
0.7
0.9-


99.9 41.8 43.4
13.7 15.6
12.5 3.4 4.5 6.8 62.5 26.5
24.0 21.8


2.0 5.8 2.6
1.2 1.5


94.5 82.8-113.2 41.1 38.2- 44.5 2.3 1.9- 2.7


8.92
6.61 3.25

1.33

1.43 0.39
0.44 0.13
0.13 0.11 0.05
0.06 0.14
1.45 0.34
0.41 0.33


0.02 0.12 0.05
0.01 0.01


0.74
0.22


63.10
46.76
22.99


12.9
13.8 17.0


9.41 14.8


10.16 2.79
3.12 0.94 0.97 0.84
0.41 0.44 1.00
10.26 2.43 2.94 2.35


0.20 0.85 0.36 0.09 0.11


12.9 7.8 8.7 8.9 7.0 8.4 19.3
12.9 20.3 31.9 11.0
17.9 15.4


14.2
23.1 22.1 9.8 8.7


5.20 5.5 1.50 3.8


0.01 0.13 5.8










middle of metacorpus: 43.8 um; middle of metacorpus to head end: 71.9 pm; metacorpus width: 30.5 Vm; metacorpus length: 33.6 um; metacorpus valve width: 9.5 um; metacorpus valve length: 12.2 um; stylet: 10.9 Vm; stylet knobs height: 2.1 um; stylet knobs width: 3.2 um; DEGO: 4.5 pm; a ratio: 1.37; body length/neck length: 3.17; stylet knobs width/height: 1.52; metacorpus length/width: 1.10; metacorpus valve length/width: 1. 28. Female as in general description. Perineal region not visible.

Description (Figs. 4, 5, 6, 7, 8, 9, 10, 11). Body pearly white, with body length (excluding neck)/maximum body width (ratio a) with an average value of 1.4 and a range of 1 to 2. Distinct posterior protuberance present (Figs. 4, 5). Neck inserts on the ventral side of body, its position varying from approximately even with anterior end of body to about one-third of body length ventrad to this point. Center line of neck and axis of body (straight line from middle of perineal area to the anterior most part of body) making an angle that varies between 20 and 1300. Cuticle distinctly annulated, often with incomplete annulations in the head and neck regions. Head region offset from body. In SEM (Fig. 6A-D) the labial disc appears slightly elevated, with the rounded and relatively large prestoma located in the middle. The labial disc and the medial lips form an anchor-shaped structure, with the ventral lip (determined from the position of the excretory pore) being pointed. In a few cases the ventral lip is not pointed, but the anchor-shaped structure is still recognizable (Fig. 6D). Inner labial sensillae are difficult to see. Head region appears as a single annule, often marked by longitudinal lines. Amphid openings are clearly distinct, rectangular. Lateral lips are arched, slightly larger than the labial disc. Cephalic framework has lateral sectors larger than ventral or dorsal sectors.



































E
0 0n


Fig. 4. Outlines of females of Meloidogyne salasi sp. n..


(50 CI(j


U



















































Fig. 5. Female of Meloidogyne salasi sp. n..

































Fig. 6. Scanning electron photomicrographs of face views of
females of Meloidogyne salasi sp. n..
























C*4 U.1 41.7










rh










The vestibule and vestibule extension are clearly distinct when observed with the LM (Fig. 7). Stylet is delicate, and the cone is usually straight, with a triangular base about 1/4 of its length, tapering to a fine, pointed tip. Opening of stylet is near the tip, in the anterior 1/4 of the cone. The shaft has approximately the same diameter throughout and is shorter than the cone. Stylet knobs offset from the shaft, and are ovoid to almost triangular in shape. Lumen of stylet in the stylet knobs is about the same as in the procorpus, but it narrows sharply in the stylet cone. Outlet of the dorsal esophageal gland is branched twice, with dorsal ampulla relatively large. Excretory pore position is relatively variable, about 1-1 times the stylet length behind the stylet knobs in 66% of the specimens observed. In a few females (4%) the excretory pore was about 1/2 stylet length behind the stylet knobs, whereas in others (6%) it was about 3 times the stylet length behind the stylet knobs. Lumen of esophagus is strongly sclerotized in the procorpus and metacorpus, but difficult to see beyond the latter. Metacorpus is relatively large and rounded (Fig. 8), with a strong, oval central valve. Esophageal glands appear as a massive, globose structure with five nucleated lobes, which are often difficult to distinguish. Nuclei are difficult to observe with bright field illumination but distinct with Nomarski differential interference optics. Perineal pattern is oval-shaped (Figs. 9, 10, 11) and has fine outer striae and somewhat coarse striae in the inner portion. The striae are mostly unbroken, smooth, relatively few in number and far apart. Perineum has no or only one striae, and only a few in the roughly circular central area of the pattern. Vulva is a transverse, smooth slit, with no or few striae coming out of its sides. Phasmids are small,



















































Fig. 7. Cephalic region of a female of Meloidogyne
salasi sp. n..




























































Fig. 8. Anterior region of a female Meloidogyne
salasi sp. n..


































Fig. 9. Photomicrographs of female perineal patterns of
Meloidogyne salasi sp. n.. A, B and C from Costa Rica.
D from Panama.
























2 5,qm


Fig. 10. Perineal patterns of Meloidogyne salasi
sp. n.. A, B and C from Costa Rica.
D from Panama.

















































Fig. 11. Scanning electron photomicrographs of
female perineal pattern of Meloidogyne
salasi sp. n..









closely spaced. Dorsal arch is high and wide, usually rectangular in shape, but somewhat square in some specimens. There is no evidence of lateral lines or interrupted striae. Tail tip is prominent in freshly mounted perineals. Punctations are lacking.

Males. Measurements of 50 males in distilled water are presented in Table 8.

Measurements of allotype in glycerin. Body length: 1,711 pm;

maximum body width: 35.3 um; body width at base of knobs: 16.6 um; body width at excretory pore; 26.6 pm; body width at middle of metacorpus: 22.2 um; excretory pore to head end: 136.7 um; middle of metacorpus to head end: 94.5 um; head height: 5.3 pm; head width: 10.9 um; excretory pore to middle of metacorpus: 48.4 jm; esophageal lobe end to head end: 243.2 pm; stylet: 19 jm; stylet base to head end: 21.8 um; stylet shaft plus knobs: 9 um; stylet cone: 10 um; stylet knobs height: 2.3 um; stylet knobs width: 3.3 um; DEGO: 4.4 jm; metacorpus width: 11.9 Um; metacorpus valve width: 3.4 um; metacorpus valve length: 8.4 um; testis: 1,034 um; testis %: 60.4; spicules: 28.8 pm; gubernaculum: 9 vm; tail length: 13.8 jm; cloaca-phasmids: 8.4 jm; ratio a: 48.4; ratio b: 7.0; ratio c: 123.9.

Description (Figs. 12, 13, 14). Vermiform, with variable body length, tapering at the anterior end (Figs. 12A, 12B) and relatively rounded at the posterior end (Figs. 12C, 12D). Head region slightly offset from body, bearing a variable number of incomplete annulations, with distinct head cap (Figs. 12A,B, 13A,B). In SEM the large, rounded labial disc is slightly elevated above the medial lips, with lateral edges slightly arcuate (Figs. 14A,B). The oval prestoma is in the center of the labial disc, encircled by six inner labial sensillae which have










Table 8. Measurements of 50 males of Meloidogyne salasi sp. n. from rice,
cv. C.R.1113.



Standard Standard error of deviCharacter Mean Range the mean ation CV (%)


Linear measurements (vm) Total length Maximum body width Body width at base of knobs Body width at exc. pore Body width at middle of metacorpus Exc. pore to head end Middle of metacorpus to head end
Head height Read width Exc. pore to middle of metacorpus Stylet
Stylet base to head end Stylet shaft + knobs Stylet cone Stylet knobs height Stylet knobs width DEGO
Metacorpus width Metacorpus valve width Metacorpus valve length Testis
Spicules
Gubernaculum Tail length Cloaca-phasmids Phasmids-tail end


1,619.0
33.9 16.8 26.8


992.0-2,093
25.4- 41.8 11.8- 20.7 23.1- 34.4


23.6 20.1- 27.0 156.9 88.0-227.0


101.7
4.5 10.4

55.8 18.2
20.6 10.4 7.7 3.1 4.6 4.1 12.6 5.1 6.8
887.1 25.8
7.8
13.0 4.1 8.8


64.0-134.0 2.5- 5.6
7.5- 13.1

18.7- 99.9
12.1- 21.8 15.9- 23.1 6.8- 12.5 4.3- 10.3 2.1- 4.2
3.5- 7.5
2.8- 5.9
8.4- 16.2 3.1- 7.1
4.8- 8.7
353.0-1,250 17.5- 34.5
5.6- 11.8 6.5- 39.0 0.3- 9.9
4.0- 17.5


Ratios


Body length/middle of metacorpus to head end Head region width/height Stylet knobs width/height Metacorpus valve length/width


Percentages Excretory pore Testis


47.5 31.8- 58.1 132.8 46.6-254.7


16.0
2.3 1.5 1.3


11.7
1.8
1.0
0.7-


21.6 3.0 3.0 2.3


9.7 6.5- 12.7 55.0 32.0- 71.6


40.87 0.49 0.19 0.33

0.22 4.56

2.61 0.09 0.15

2.89 0.31 0.27 0.20 0.17 0.07 0.09 0.10 0.24 0.12 0.14 25.24 0.63 0.19 0.65 0.35 0.36


0.92 5.46

0.32 0.03 0.04 0.04


0.20 1.11


289.04
3.53 1.40 2.40


17.8
10.3 8.3 8.9


1.58 6.6
32.31 20.5


18.51 0.69 1.12

20.44 2.19 1.96
1.43 1.22 0.54 0.66
0.72 1.73 0.88 0.99
178.51 4.52 1.34 4.66 2.50 2.58


18.2
15.3 10.7

36.6
12.0 9.5
13.7 15.7 17.6 14.1 17.4 13.7 17.2 14.4 20.1 17.4 17.0 35.7 59.6 29.1


6.51 13.6 38.62 29.0


2.33 0.26 0.32 0.30


14.4 11.4
21.2 21.7


1.43 14.7 7.90 14.3



















0 0
E r,
i/ U/I,













0 0






04 0
0S
Io0

0

0
0 E


~o







Fig. 12. Males of Meloidogyne salasi sp. n.. A Esophageal region
(ventral). B Cephalic region (lateral). C,D Tail (lateral).


































Fig. 13. Anterior region of males of Meloidogyne salasi sp. n..
A,B Scanning electron microscope photomicrographs.
C,D Light microscope photomicrographs.




































Fig. 14. Scanning electron photomicrographs of males of Meloidogyne
salasi sp. n.. A,B Face views. C Lateral field.
D Tip of spicules showing pores.





2R90'3 20KU 5LIJ










pitlike openings. Stoma has a slitlike opening. Medial lips are wider than the labial disc, forming a continuous head cap with it, with no discernible indentations at the lateral junctions. Four cephalic sensillae appear as slight, small cuticular depressions on the medial lips, two on each. Amphidial openings are relatively long slits below the lateral edges of the labial disc. Lateral lips are almost inconspicuous, and marked by short grooves that start near the lateral junction of the medial lips and the labial disc, and extend into the head region. One to three rows of short, incomplete annulations are present at different levels of the head region (Figs. 13A,B). Frequently the specimens have one row on one side and two or three on the opposite side. Cuticle has distinct annules, about 1.9 Um wide near the head region, 2 pm wide around the middle of the body and 1.6 pm wide near the tail. Lateral field is about 6, 7.5 and 5 pm wide near the anterior, middle portion and tail areas of the body, respectively. There are usually four lines in the lateral field, one at each edge of the ridge and two in the inner portion, but in some specimens five or up to six lines are visible for some distance in the middle of the body; the additional lines are fainter. Lateral fields start as two lines with crenate edges near the base of the stylet, some four to 10 annules behind the head region, continue posteriorly as far as the middle of the procorpus, where the inner two lines appear, and continue to the posterior end. The tail portion is twisted about 90*. The lateral fields are areolated in their entirety, and usually correspond with the body annulations, but in some areas, especially the middle portion, they do not (Fig. 14C). The cephalic framework is sclerotized, and its lateral sectors are slightly larger than the head cap (Figs. 12A; 13C,D).









Stylet is robust, with a pointed cone, slightly longer than the shaft. The cone has the opening near the tip and a triangular base in the basal

of its length. Stylet shaft is of same diameter throughout, with ringlike structure near its base (Figs. 12A, B). Stylet knobs are rounded, offset from the shaft, with an ascending slope toward base of stylet shaft (Figs. 12B; 13C,D). Lumen of stylet is almost as wide as that of the procorpus, but narrows at the cone. Outlet of the dorsal esophageal gland has two branches, with a relatively small dorsal ampulla. Procorpus is two to three times as long as the muscular, elongated, oval metacorpus (Fig. 12A). The metacorpus has a strongly sclerotized central valve. Nerve ring encircles the short isthmus. Distinct excretory pore, with long, curved excretory duct that disappears as it approaches the intestine. Basal lobe of esophagus overlaps the intestine ventrally and has three nuclei, with the anterior nucleus near beginning of lobe and posterior nucleus near the end of the lobe. Hemizonid 1-2 annules long, located 1-2 annules anterior to excretory pore. Intestinal caecum extends anteriorly on dorsal side of body to about the level of the nerve ring. Most specimens have one outstretched testis, but it may be reflexed for a short distance. A few specimens have two testes. If two testes are present, one may be outstretched and the other reflexed, but both of about the same length. Sperm are globular, granular. Spicules are long, arcuate, typical of the genus (Figs. 12C,D). In SEM each spicular tip shows one transverse opening (Fig. 14D). Gubernaculum is simple. Phasmids are located typically below the cloacal opening, with a pore-like opening. Body twists about 90* near the cloacal region.

Second-stage juveniles. Measurements of 50 juveniles in distilled water are presented in Table 9.










Table 9. Measurements of 50 second-stage juveniles of Meloidogyne
salasi sp. n. from rice, cv. C.R.1113.



Standard Standard
error of deviCharacter Mean Range the mean ation CV (%)


Linear measurements (um) Total length Esophageal lobe base to
head end
Maximum body width Body width at excretory po Middle of metacorpus to excretory pore
Middle of metacorpus to
head end
Head region width Head region height Stylet
Stylet cone Stylet knobs width Stylet knobs height Stylet base to head end Stylet shaft DEGO
Metacorpus valve length Metacorpus valve width Excretory pore to head end Tail length Tail terminus length Tail terminus width at beginning
Anal width Anus-beginning of terminus


464.4 422-503 2.59


121.8 16.2
re 15.0


103-153 15.3-19.3 13.1-15.9


23.6 16.8-31.5


56.7 6.2
3.3 11.4 5.2
2.3 1.5
14.7 4.7 3.7 3.9 3.4 80.3
67.8 19.7

5.1 11.8
47.9


Ratios
a 28.6 b 3.8 c 6.8
Tail length/anal width 5.7 Tail length/tail terminus length 3.5 Head region width/height 1.9 Stylet knobs width/height 1.5 Metacorpus valve length/width 1.1

Percentages
Excretory pore 17.2


50.6-62.1 5.0- 7.8
1.8- 5.6
9.2-13.3
3.7- 6.8
1.5- 2.8
1.0- 2.1
12.1-16.2 2.8- 6.2
2.1- 5.3
2.8- 5.3
2.5- 4.3
71.5-89.6 56.5-80.2 11.8-26.2

3.7- 6.2
10.7-15.0
38.1-58.7


23.9-32.2 3.0- 4.4
5.9- 7.7
4.2- 6.8


2.4
1.2
0.7
0.6-


1.31 0.11
0.59

0.46

0.36 0.08 0.12 0.15 0.11
0.04 0.03 0.09
0.09 0.08 0.06
0.04 0.57 0.73 0.47

0.09 0.10
0.67


0.24
0.04 0.05
0.07

0.08
0.05 0.03
0.02


18.35

9.26 0.83 0.64


3.27 13.8


2.58 0.58 0.87 1.07 0.83 0.30 0.22
0.69 0.70 0.58 0.45 0.33 4.03 5.16 3.33

0.64 0.71 4.77


1.73 0.30 0.42 0.54

0.58 0.37 0.25
0.16


4.5 9.2
25.8 9.3
15.8
13.2 14.8 4.6 14.8
15.5 11.4 9.7 5.0 7.6 16.8

12.3 6.0 9.9


6.0 7.9 6.1 9.4

16.7
19.3 16.7
13.9


0.08 0.61 3.5


16.0-18.7





















EE


B
















LI D




K1 E


Fig. 15. Second-stage juveniles of Meloidogyne salasi sp. n..
A Esophageal region (lateral). B Cephalic region
(lateral). C Tail (dorsal). D Tail (lateral).


















































Fig. 16. Scanning electron photomicrographs of face views of secondstage juveniles of Meloidogyne salasi sp. n..


















































Fig. 17. Lateral field of second-stage juvenile
of Meloidogyne salasi sp. n..



































Fig. 18. Second-stage juveniles uf Meloidogyne salasi sp. n..
A,B Anterior region. C,D Tail terminus.







68










Description (Figs. 15, 16, 17, 18). Body is vermiform, tapering at both ends but much more so posteriorly (Figs. 15A,B,C,D). Head region slightly narrower than the body, and elevated head cap (Figs. 15B). In SEM the elongated labial disc is slightly elevated above the medial lips, with lateral edges straight or almost so (Fig. 16A). Oval prestoma in the center of the labial disc, encircled by six inner labial sensillae with pit-like openings. Stoma with a small slit-like opening. Medial lips crescentic in most specimens, wider than the labial disc, with no discernible indentations at the lateral junctions with it, forming a dumbell-shaped cap. In a few specimens one of the medial lips is pointed (Fig. 16B). Amphidial openings are slitlike, and located below the lateral edges of the labial disc. Lateral lips are narrow, with straight or slightly arcuate edges, almost parallel to the lateral edges of the labial disc. Head region is smooth, without annulations. Cephalic framework is weakly developed. Body is distinctly annulated, the annulations being discernible with the LM up to the beginning of the tail terminus. Lateral fields are areolated, with four lines, the external two are slightly crenate (Fig. 17.). Anteriorly the exterior two lines begin at about the middle of the procorpus, followed by one and finally two interior lines (Fig. 17). All four lines continue past the anus, where the central lines disappear and the two exterior ones continue for a short distance to the beginning of the tail terminus. The stylet is weakly developed, and has small, rounded knobs, one slightly larger and in a lower position than the other two (Figs. 15A,B; 18A,B). The knobs have an ascending slope toward the shaft. A ring-like structure encircles the shaft near its base (Fig. 15B). Ampulla of the dorsal gland duct near its opening into the lumen of the esophagus.










Procorpus is about 2-2 times as long as the muscular, oval metacorpus which has a sclerotized central valve. Nerve ring encircles the narrow isthmus. Hemizonid located 1-2 annules anterior to the excretory pore, about 1 annule long. Excretory pore located at about the same level or slightly posterior to the nerve ring. The curved excretory duct disappears as it approaches the intestine. Basal lobe of esophagus rather short, with three nuclei, the anterior one located near its beginning and the posterior one near its end. The basal esophageal lobe overlaps the intestine ventrally (Fig. 15A). Anal opening is a small pore on the cuticle. Rectum is weakly dilated. Tail relatively long, tapering to a fine, rounded, slightly clavate terminus (Figs. 15C,D).

Eggs. Measurements of 50 eggs in distilled water are presented in Table 7.

Description. Eggs similar to those of other species of the genus, and are enclosed in a soft, highly water-soluble gelatinous matrix. Up to 2,000 eggs/egg mass have been counted on galled rice roots collected from the type locality (L. Salazar, personal communication). Diagnosis: M. salasi sp. n. is closely related to the recently described M. kralli (Jepson, 1983), and also to M. acronea (Coetzee, 1956) and to M. graminis (Sledge and Golden, 1964).

M. salasi sp. n. can be distinguished from M. kralli by the dimensions of the female (body length of 486 pm vs 463 um, maximum body width of 338 um vs 306 um), the straight shorter stylet (10 Um vs 13.1 pm), longer excretory pore of the female (32 pm vs 15.8 pm), by the higher dorsal arch of the perineal pattern, and absence of a postero-laterally directed irregular double incisure on either side of the tail region of the perineal pattern, longer males (1,619 pm vs 1,076 um), greater a and










c ratios in the males (47.5 and 132.8 vs 31.7 and 117, respectively), shorter stylet cone in the male (7.7 um vs 9.5 pm), longer excretory pore in the males (156.9 um vs 127 um), areolation of the lateral fields, annulations in the head region of the male (up to 3 vs 1), position of the phasmids on the male (posterior to cloaca vs at level of cloaca). Additional differentiating characters in the second-stage juveniles include the body length (464 um vs 439 pm), the smaller a and b ratios (28.6 and 3.8 vs 31 and 6.5, respectively) and the smaller tail/anal width ratio (5.7 vs 7).

M. salasi sp. n. can be distinguished from M. acronea by the female body length (486 Um vs 980-1,040 um), maximum body width (338 um vs 530750 pm), shorter stylet in the female (10 um vs 12 um), shorter spicules of the male (25.8 Um vs 33-35 Um), longer phasmids-tail end distance (8.8 um vs 4 pm), areolation of the lateral fields in the male, shorter second-stage juveniles (464 um vs 490 um), smaller a, b, and c ratios in the juveniles (28.6, 3.8, and 6.9 vs 32, 5.4, and 9.4, respectively), longer tail of the juveniles (67.8 pm vs 49 pm) and longer tail terminus (19.7 Pm vs 3.5 um). Finally, M. salasi sp. n. can be distinguished from M. graminis by the body length of the female, maximum body width and DEGO (486, 338, and 4.9 um vs 726, 472, and 3.7 pm), respectively), the absence of lateral lines in the perineal pattern, the fine striae in the perineal pattern; in the males by the greater a ratio (47.5 vs 43.5), the smaller c ratio (132 vs 187), the longer DEGO (4.1 Um vs 2.4 pm), the longer tail (13 um vs 8.4 Um) and the areolation of the lateral fields; in the second-stage juveniles by the length (464 pm vs 475 um), the smaller a ratio (28.6 vs 31.7), the greater b ratio (3.8 vs 2.3), the longer DEGO (3.7 um vs 2.4 um), the shorter esophageal lobe to head end distance










(121.8 um vs 200 pm), the shorter tail (67.8 pm vs 78 Um) and the greater tail/anal width ratio (5.7 vs 4.3).

Host range: M. salasi sp. n. did not infect any of the plant species used in the North Carolina Differential Host test (Taylor and Sasser, 1978). Greenhouse studies conducted in Panama (Tarte, 1981) showed that Cynodon plectostachyus, C. dactylon, Ischaemum ciliare, Digitaria decumbens, Tripsacum laxum, Echinocloa polystachya, Leucaena leucocephala, Kazungula sp., Brachiaria ruziziensis, B. zuazilandensis, B. rugulosa, Panicum maximum and Saccharum sinensis are poor hosts for this nematode. Figueroa (1973) reported that Homolepis aturensis is a host. The grass Echinocloa colonum is a host under field conditions at the type locality (R. Lopez, unpublished data).

Holotype (female): Isolated from greenhouse culture derived from original population obtained at La Cuesta, Costa Rica. Slide M-39, Nematode collection, Laboratorio de Nematologia, Facultad de Agronomia, Universidad de Costa Rica, San Jose, Costa Rica. Allotype (male): Same data as holotype. Slide M-13, Laboratorio de Nematologia, Facultad de Agronomia, Universidad de Costa Rica, San Jose, Costa Rica.

Paratypes (males, females and second-stage juveniles): Same data as holotype. USDA Nematode collection, Beltsville, Maryland. Type host and locality: Rice (Oryza sativa L.), cv. C.R. 1113, from La Cuesta, province of Puntarenas, Costa Rica.
















CHAPTER IV
VARIABILITY OF Meloidogyne incognita FROM FLORIDA Introduction

Prior to 1949 several investigators reported variations in populations of root-knot nematodes [then known as Heterodera marioni (Cornu, 1979) Goodey, 1932] parasitizing different plants. The first such record is apparently that of Sherbakoff (1939). He described root-knot disease on upland cotton growing on land where the previous crop was cotton. But he noted that in another field upland cotton was not infected when the previous crop was tomato infected with root-knot nematodes. Variation in host specificity on peach to root-knot nematode populations were reported by several authors (Day and Tufts, 1940; Christie and Havis, 1943). Evidence of the existence of physiological variation within H. marioni was also given in other reports (Christie, 1946; Christie and Alvin, 1944; Reynolds, 1949).

In 1949, the genus Meloidogyne was reestablished and separated from Heterodera Schmidt, 1971 (Chitwood, 1949). Five species and one variety were described, viz. M. exigua, M. javanica, M. incognita, M. hapla, M. arenaria and M. incognita var. acrita.

Sasser (1954) devised a scheme for the identification of the five species and one variety described by Chitwood (1949) based on the ability of each to infect certain plant species. This method was successful in identifying species that occurred in a given region, but it was not reliable when the species came from different geographical regions









(Sasser, 1966; Sasser, 1972). In these instances the differential hosts may not give what has been called the "usual response for each species" (Sasser, 1972).

Results of several hundred differential host tests on populations from all over the world were used as the basis to separate four host races within M. incognita (Sasser, 1979; Sasser and Carter, 1982). These race designations are based on the ability to infect and reproduce on cotton, cv. Deltapine 16, and tobacco, cv. NC-95. Race 1 does not parasitize or reproduce on either, whereas race 2 reproduces readily on tobacco. Race 3 reproduces on cotton but not on tobacco, and race 4 reproduces on both. According to Sasser and Carter (1982), the consistency of host response to the four races, the widespread occurrence, especially of races 1, 2 and 3, and the number of populations involved shows that these races are stable taxa. The four races are as yet indistinguishable morphologically and are apparently unrelated to the cytological races distinguished in this species (Triantaphyllou, 1981).

Schemes other than that of Sasser (1979) have been used to distinguish races of M. incognita. The degree of reproduction on soybean and alfalfa varieties was used by Boquet et al. (1975) and by Goplen et al. (1959). Other authors used the amount of root necrosis, root galling and the capability to parasitize sweetpotato varieties to distinguish races of M. incognita and M. incognita var. acrita (Giamalva et al., 1963; Martin and Birchfield, 1973).

Fox and Miller (1973) distinguished two races of M. incognita by

the number of egg masses produced on five differential hosts. They concluded that the number of galls was not a reliable index of the reproduction of root-knot nematodes.










Populations of M. incognita in Tennessee differ considerably in

pathogenicity (Southard and Priest, 1973). Six races were distinguished among 17 isolates evaluated on six differential hosts.

In Florida, Perry and Zeikus (1972) found variation in the response of four populations of M. incognita to differential hosts. One population collected from strawberry reproduced well on this host, whereas another population collected from tomato was the only one that reproduced on pepper, cv. California Wonder. A third population, collected from sugarcane, reproduced on tobacco, cv. NC-95; the others did not reproduce on this host. Finally, a population collected from peach was the only one to reproduce on 'Okinawa' peach rootstock.

Kirby (1972) reported variation in host preference among 14 populations of M. incognita collected from nine counties in Florida. Some populations caused severe galling on cotton, cv. Coker 201, whereas most did not. Variations in pathogenicity on sweetpotato, cv. Puerto Rico, also were noted. Only one population caused galling on tobacco, cv. NC-95.

More recently, Lopez and Dickson (1977) found no differences in

host reactions among three populations of M. incognita from Florida; all were identified as race 1.

When Chitwood (1949) reestablished the genus Meloidogyne, he used

the characteristics of the female perineal pattern, the stylet of secondstage juveniles, males and females, and the location of the dorsal esophageal gland orifice to distinguish the five species and one variety he described.

Dropkin (1953) concluded that the general shape and perhaps other characters of the perineal pattern are under hereditary control.










Variability in populations originating from a single juvenile of M. incognita var. acrita was less than in populations with a mixed ancestry.

Allen (1952) demonstrated the variability in perineal patterns of M. incognita var. acrita originating from single egg mass isolates and grown on three plant species.

Triantaphyllou and Sasser (1960) observed that in many isolates of M. incognita var. acrita the perineal patterns varied from typical acrita to typical incognita type. They stated that the morphological distinction between the subspecies was often uncertain, and that since the perineal pattern gives little information on the behavior of a given population, the division of the species into two subspecies had no practical purpose. They suggested that all populations with perineal patterns ranging from incognita to acrita-type be considered one species, M. incognita. Esser et al. (1976) however, considered that some differences in the coarseness of the striae in the perineal pattern, the dilation of the rectum, the a and c ratios of second-stage juveniles and the spicules of the male were solid enough features to give M. acrita the status of species.

Riggs and Winstead (1959) were able to correlate the ability of

certain isolates of M. incognita var. acrita and M. incognita to parasitize resistant tomatoes with certain morphological characters, i.e., patterns with more arch and longer second-stage juveniles. These differences, however, were not enough to exclude any of the new strains from the parental type. Priest and Southards (1971) could not morphologically distinguish six races of M. incognita among 16 populations of this species. Significant differences were noted in total length,










c ratio and stylet length of second-stage juveniles among certain populations.

Kirby (1972) found variation among 14 populations of M. incognita from Florida in mean vulva length, female interphasmidial and anus-vulva distances, DEGO of males and total length, stylet base to head end, maximum body width, tail length, DEGO and a and c ratios of second-stage juveniles. Range values of all measurements overlapped among populations. Lopez and Dickson (1977) reported statistically significant differences among three populations of M. incognita race 1 in several morphometric parameters. These parameters were stylet and DEGO of females, stylet, DEGO and spicules (chord of arch) of males, and total length, stylet base to head end, tail length, maximum body width, anal width and a and c ratios of second-stage juveniles. In spite of these differences, no individual character could be used to distinguish among the three populations since the ranges of all measurements overlapped.

This study was conducted to characterize three populations of M. incognita from Florida by morphology and the reaction of differential plants.

Materials and Methods

Nematode Populations

The designations and original sources of three Florida populations of M. incognita were: M-165, tobacco, Alachua County, 1974; M-195, corn, Suwannee County, 1977, and M-198, tomato, Manatee County, 1980. From several dozen egg masses these populations were increased on tomato, cv. Rutgers, in a greenhouse. A mixture of soil and builders sand (3:1 v/v, pH 7.1, 1.5% O.M., 91.8:6.2:2.0% sand:silt:clay) treated with steam at










100*C for 24 hours was used in all tests. Each pot was fertilized twice a week with 100 ml of a 1% solution of Nutrisol (12-10-20). Differential Plants

Seven differential plants were inoculated with each population, and the plant responses were evaluated 60 days later. The differential plants used were identical to those used in Costa Rica: corn, cv. Minnesota A-401, tobacco, cv. NC-95, pepper, cv. California Wonder, cotton, cv. Deltapine 16, peanut, cv. Florunner, watermelon, cv. Charleston Grey, and tomato, cv. Rutgers.

Pregerminated seeds of all plants, except tobacco, were prepared. Plants were inoculated at the following ages: corn-10 days old; peanut, cotton and watermelon-15 days old; tomato-30 days old, and tobacco and pepper-50 days old. The inoculum consisted of eggs and a few secondstage juveniles that were collected using a 1.05% sodium hypochlorite solution (Hussey and Barker, 1973).

Approximately 10,000 eggs and or second-stage juveniles were

pipetted over 1,000 cm3 of soil in clay pots. Individual seedlings were planted and 500 cm3 of soil added. Four replications of each differential plant-population combination were placed randomly on a greenhouse bench and kept separated by plastic dividers. The pots were fertilized twice a week with 100 ml of 1% Nutrisol fertilizer solution (12-10-20) during the first five weeks of plant growth.

Sixty days after inoculation the root systems were removed and immersed in a 0.0016% Phloxine B solution for one hour (Dickson and Struble, 1965) to stain the egg masses. Each root system was rated using the following scale (Taylor and Sasser, 1978):










0: 0 egg masses 3: 11-30 egg masses 1: 1-2 egg masses 4: 31-100 egg masses

2: 3-10 egg masses 5: more than 100 egg masses.

Perineal patterns were prepared whenever it was judged appropriate to verify the identity of the females reproducing on a particular differential plant.

Morphology

For each character, 20 specimens in each population were observed. Al measurements were taken from outlines drawn with a camera lucida at 1,000X magnifications, except second-stage juvenile length (100X) and female length and maximum body width (40X).

Females were dissected from galled tomato roots boiled in lactophenol for two minutes. The perineal patterns were prepared according to the technique developed by Franklin (1962) and modified by Taylor and Netscher (1974). The perineals were divided into zones, following the method of Esser et al. (1976).

Whole females were mounted in lactophenol on a cavity slide and their length and maximum body width drawn. The females were removed from the solution and punctured near mid-body with a fine needle to release the internal pressure. The neck and head regions were excised and mounted in a drop of lactophenol on a glass slide, covered with a coverslip and ringed with Zut.

To obtain second-stage juveniles, egg masses were collected randomly from galled tomato roots and kept for 18-24 hours in distilled water. Live juveniles were mounted in a drop of water on a glass slide ringed with Zut and covered with a coverslip. Fifteen to 20 minutes later the juveniles were observed under a light compound microscope,










certain characters were outlined and the position of the hemizonid in relation to the excretory pore and the dilation of the rectum were noted and recorded.

To obtain males, galled tomato roots were dissected in petri dishes containing distilled water. After one to two hours the males were mounted using the same technique as described for the juveniles.

Results

Differential Plants

Populations M-195, M-165 and M-198 were identified as M. incognita race 1, race 2 and race 3, respectively (Table 10). Tomato, cv. Rutgers, pepper, cv. California Wonder, watermelon, cv. Charleston Grey, and corn, cv. Minnesota A-401, were good hosts for all M. incognita populations. Peanut, cv. Florunner, was not a host for any population. Morphology

The interpretation of the predominant type of perineal pattern for each population is presented in Table 11. None of the populations had a posterior protuberance, lateral incisures or striae in the perineum. A few striae on the vulva of population M-165 were observed coming out of its sides, but no such striae were present in M-195 or M-198. The three populations had relatively few striae in zone 1. The striae of populations M-195 and M-198 in zones 2, 3 and 4 were wavy, broken and relatively few in number, whereas those of M-165 were wavy and broken, but relatively moderate in number. The general shape of the perineal patterns in the three populations was pyriform, with a high trapezoidal dorsal arch. Some slight variants from this predominant shape were observed. An illustration of one perineal pattern from each population is presented in Fig. 19.









Table 10.


Response of seven differential plants to three populations of Meloidogyne incognita from Florida.


Population
Plant M-195 M-165 M-198

,
Tomato 'Rutgers' 5 5 5 Tobacco 'NC-95' 0 5 1 Pepper 'California Wonder' 5 5 5 Cotton 'Deltapine 16' 0 0 5 Peanut 'Florunner' 0 0 0 Watermelon
'Charleston Grey' 5 5 5 Corn 'Minnesota A-401' 4.5 4.5 4.5 race 1 race 2 race 3

*
Average of four replicates. Response evaluated according to the number of egg masses/root system. 0 = 1; 1 = 1-2;
2 = 3-10;3 = 11-30; 4 = 31-100 and 5 = >100 egg masses.










Interpretation of the predominant type of pattern of females of three host races of incognita from Florida.


perineal Meloidogyne


Population
Character M-195 M-165 M-198 race 1 race 2 race 3

*
Posterior protuberance A A A Vulva lip striae A F A Perineum A A A Lateral incisures A A A

Striae
Zone 1 F F F Zone 2 FWB MWB FWB Zone 3 FWB MWB FWB Zone 4 FWB MWB FWB

Shape pyriform, with a high trapezoidal dorsal arch.

,
A = absent; F = Few; M = moderate in numbers; W = wavy; B = broken.


Table 11.

















































Fig. 19. Photomicrographs of female perineal patterns
of three populations of Meloidogyne incognita
from Florida.










The average values of the female morphometric characters are presented in Table 12. No significant differences in excretory pore, stylet, anus-vulva and interphasmidial distances were found among populations. Population M-195 had a significantly shorter DEGO than M-165 and M-198, whereas the latter had a significantly shorter body length than M-195 and M-165. The maximum body width of M-165 was significantly greater than those of M-195 and M-198. The vulva of M-165 was significantly longer than that of M-198. The range of these measurements are presented in Table 18 of the appendix.

The mean values of the characters of second-stage juveniles are

presented in Table 13, and the ranges in Table 19 of the appendix. No significant differences among populations were found in maximum body width, anal width and stylet base to head end. Population M-198 had significantly shorter total length and tail length than populations M-165 and M-195. This last population had significantly longer DEGO than the other two. The difference in DEGO between M-165 and M-198 was significant also. The a ratio was significantly different for each population. The c ratio of M-198 was significantly greater than those of M-165 and M-195. The second-stage juveniles of the three populations had dilated recta, and the hemizonid was located anteriorly to the excretory pore.

Morphological observations and the average values of some morphometric characters of the males are presented in Table 14. Range values for these characters are presented in Table 20 in the appendix. All males observed had four lateral lines, areolated lateral fields, and in all populations 95% of them had one gonad. No significant differences










Table 12. Comparative morphological data (pm) from
host races of Meloidogyne incognita from


females of three Florida.


Population
Character M-195 M-165 M-198 CV (%) race 1 race 2 race 3


Excretory pore 26.0 a 23.8 a 27.4 a 35.0 Stylet 16.5 a 16.0 a 16.8 a 7.1
**
DEGO 4.3 b 4.9 a 5.1 a 17.4 Maximum body width 441.0 a 535.0 a 463.0 b 12.0 Body length 716.0 a 715.0 a 641.0 b 12.8 Vulva slit length 21.9 ab 22.7 a 20.9 b 8.5 Anus-vulva 16.1 a 16.3 a 15.6 a 9.2 Interphasmidial distance 18.3 a 19.7 a 18.2 a 15.3

*
Mean of 20 observations. Means in horizontal rows followed by the same letter do not differ significantly according to Duncan's Multiple Range
Test (P = 0.01).
**
DEGO refers to the distance between the base of the stylet knobs and
the dorsal esophageal gland orifice.










Table 13. Comparative morphological data (um) from second-stage
juveniles of three host races of Meloidogyne incognita
from Florida.



Population
Character M-195 M-165 M-198 CV (%) race 1 race 2 race 3

*
Total length 430.0 a 431.0 a 399.0 b 3.5 Tail length 54.0 a 55.4 a 43.0 b 5.8 Maximum body width 15.0 a 15.4 a 15.1 a 4.0 Anal width 10.5 a 10.8 a 10.7 a 5.6 Stylet base to head end 15.3 a 15.2 a 15.1 a 2.9
**
DEGO 2.4 b 2.9 a 3.1 c 9.6 a 28.8 b 27.9 a 26.2 c 4.5 c 8.0 a 7.7 a 9.2 b 5.8 Rectum dilation yes yes yes Hemizonid anterior anterior anterior


Mean of 20 observations. Means in horizontal rows followed by the
same letter do not differ significantly according to Duncan's Multiple
Range Test (P = 0.01).
**
DEGO refers to the distance between the base of the stylet knobs and
the dorsal esophageal gland orifice.

Position in relation to the excretory pore.










Comparative morphological data races of Meloidogyne incognita


(pm) from males of three host from Florida.


Population
Character M-195 M-165 M-198 CV (%) race 1 race 2 race 3


Stylet 23.1 a 23.6 a 23.4 a 5.6 Spicules (chord of arch) 28.5 a 31.0 a 29.9 a 14.0 DEGO 2.9 b 3.2 ab 3.4 a 16.6 Areolation yes yes yes Number of lateral lines 4 4 4 % males with one gonad 95 95 95


*
Mean of 20 observations. same letter do not differ Range Test (P = 0.01).


Means in horizontal rows followed by the significantly according to Duncan's Multiple


At
DEGO refers to the distance between the base of the stylet knobs and the dorsal esophageal gland orifice.


The rest of the males had two gonads.


Table 14.










among populations were found in stylet and spicules (chord of arch). Population M-195 had a significantly shorter DEGO than M-198.

Ranges of all measurements and ratios for females, second-stage juveniles and males overlapped among the three populations.

Discussion

The finding of race 1, 2, and 3 of M. incognita confirmed previous reports (Araujo et al., 1983; Kirby, 1972; Lopez and Dickson, 1977) that at least three host races of M. incognita exist within the state of Florida. However, there was a small difference in the reaction of one of the differential plants. Corn was rated as a poor host for the M. incognita populations studied by Kirby (1972) and Lopez and Dickson (1977), but a good host for the populations used in this study. Difference in the corn cultivar and the M. incognita populations used in these studies, as well as in the rating scales used to evaluate the response of this differential plant could explain this difference.

When differential plant responses were compared to those of the Costa Rican populations, no differences were noted, except that race 3 of M. incognita was not detected from Costa Rica.

Several statistically significant differences among the three populations studied were found in certain morphometric characters. It could be postulated that there is a relationship between some of these characters and the ability of a population to parasitize a given differential host. For instance, one could associate host race 3 with a shorter female body length and vulva, and a shorter second-stage juvenile with shorter tails, smaller a ratio and greater c ratio and DEGO values. Race 2 could be associated with a larger female body width, and intermediate DEGO and a ratio values in the second-stage juvenile. Race 1










could be associated with a shorter DEGO in the females, second-stage juveniles and males, and a greater c ratio in the second-stage juveniles. This association, however, has a weak basis as the ranges of all measurements and ratios overlapped among populations, making it difficult to distinguish them. A somewhat similar situation was found by Hirschmann (1981). She analyzed the external morphology of second-stage juveniles and males of 14 populations of M. incognita belonging to two (A and B) cytological races with the SEM. All four host races were represented among the populations studied. No correlation between morphology and host race was found. Some populations in each cytological race appeared to be distinct, but still shared the general features characteristic of the species.

When the morphometric data gathered in this study were compared to those of previous studies in Florida, some similarities and differences were noted. The general shape of the perineal patterns, the characteristics of their striae, the absence of posterior protuberance, lateral incisures and striae in the perineum and vulva length of the females were similar to the reports by Kirby (1972) and Lopez and Dickson (1977). Similarities were found also in stylet base to head end, DEGO, maximum body width, anal width, rectum dilation and anterior location of the hemizonid in relation to the excretory pore in second-stage juveniles, and in areolation, number of lateral lines, stylet and DEGO of males. The a and c ratios, and the tail length of second-stage juveniles had similar values, in most cases, to those reported earlier, except that M-198 had a smaller a ratio, a shorter tail and a greater c ratio.

Differences observed included slightly shorter interphasmidial and anus-vulva distances in the females, longer female DEGO, slightly longer









female stylet and maximum body width of second-stage juveniles. In M-195 and M-198 the spicules (chord of arch) were shorter, and in M-195 and M-165 the second-stage juveniles were longer, whereas they were shorter in M-198, as compared to the previous reports of populations of this species from Florida (Kirby, 1972; Lopez and Dickson, 1977).

As in previous comparisons, some differences and some similarities were found when the data from the Floridian populations were compared to M. incognita populations from Costa Rica. The male spicules (chord of arch), as well as the female interphasmidial distance, had smaller values than the populations from Costa Rica. The female stylets were slightly longer in the Floridian populations, whereas the vulva and the anusvulva distance were slightly shorter.

Similarities included the absence of a posterior protuberance in the female body, as well as the lack of vulva lip striae, perineum striae and lateral incisures in the perineal patterns, the presence of a few striae in zone 1, the character of the striae in zones 2, 3, and 4 of the perineals, the excretory pore and the maximum body width and body length of the females. The DEGO of the females were also similar, except that in M-198 it was slightly longer. In males, the DEGO, stylet, areolation of the lateral fields and number of lateral lines were also concordant to those from Costa Rica. The maximum body width, anal width, stylet base to head end, anterior location of the hemizonid with respect to the excretory pore and rectal dilation of the second-stage juveniles were also similar to those of the Costa Rican populations. The DEGO was similar in M-165 and M-198 but slightly shorter in M-195. The tail was shorter in M-198 and similar in the other two populations. The a ratio was greater in M-198 than in the populations from Costa Rica. Finally,




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DIFFERENTIAL PLANT RESPONSES, MORPHOMETRICS AND ELECTROPHORETIC PATTERNS OF SOME Meloidogyne spp. FROM COSTA RICA AND FLORIDA, U.S.A., AND THE DESCRIPTION OF Meloidogyne salasi sp. n. By ROGER LOPEZ CHAVES A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1984

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This dissertation is dedicated first of all to the gentleman who inspired me in the study of the science of nematology, the distinguished Costa Rican scientist Professor Luis Angel Salas Fonseca. Thank you, Don Luis, for letting me drink from the stream of your vast knowledge for so many years, and for giving me the opportunity to meet a living example of a wonderful human being. The work is also dedicated to my parents, Roger Lopez C. and Francisca Chaves M. , to my wife Ana I, Chaverri M. and to our children, Susana Maria, Roberto Enrique, Jose Francisco and Juan Pablo.

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ACKNOWLEDGMENTS The facilities provided by Dr. D.W. Dickson, chairman of the supervisory committee, are greatly appreciated. Gratitude is extended to Dr. G.C. Smart, Jr., Dr. R.E. Stall, Dr. R.A. Dunn, and Dr. J.R. Rich for serving as members of the supervisory committee. The help provided by Mr. F.E. Woods and Mr. R.A. Henn in reviewing and correcting the first manuscript, and Mr. A.L. Taylor with the photographic work is acknowledged also. Special thanks are given to my colleague, Ing. Agr. Luis Alejandro Salazar Figueroa, for his invaluable help during several years, and to all the personnel at the Laboratorio de Nematologia, Facultad de Agronomia, Universidad de Costa Rica. The patience and understanding of my wife Ana Isabel and our children were vital for the success of this investigation. The financial support of the Consejo Nacional de Investigaciones Cientificas y Tecnologicas is deeply appreciated, as well as the assistance provided by the Universidad de Costa Rica. iii

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TABLE OF CONTENTS Page DEDICATION ii ACKNOWLEDGMENTS iii LIST OF TABLES v LIST OF FIGURES vii ABSTRACT ix CHAPTER I INTRODUCTION 1 II VARIABILITY OF Meloidogyne spp. FROM COSTA RICA 4 Introduction 4 Materials and Methods 11 Results 16 Discussion 30 III DESCRIPTION OF Meloidogyne salasi sp. n 37 Introduction 37 Materials and Methods 38 Species Description 40 IV VARIABILITY OF Meloidogyne incognita FROM FLORIDA 73 Introduction 73 Materials and Methods 77 Results 80 Discussion 88 V ELECTROPHORETIC PATTERNS OF SOME Meloidogyne spp. FROM COSTA RICA AND FLORIDA 92 Introduction 92 Materials and Methods 93 Results 97 Discussion 101 VI CONCLUSIONS 104 APPENDIX 108 LITERATURE CITED 114 BIOGRAPHICAL SKETCH 12 4 iv

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LIST OF TABLES Table 1 Designations, sources and selected ecological characteristics of the collection sites of 16 populations of Meloidogyne spp. from Costa Rica 13 2 Interpretation of the predominant type of perineal pattern of females of 16 populations of Meloidogyne spp. from Costa Rica 17 3 Morphometric characters of females of 16 populations of Meloidogyne spp. from Costa Rica 22 4 Morphometric characters of second-stage juveniles of 16 populations of Meloidogyne spp. from Costa Rica 24 5 Morphological characters of males of 16 populations of Meloidogyne spp. from Costa Rica 27 6 Response of seven differential plants to 16 populations of Meloidogyne spp. from Costa Rica 29 7 Measurements of 50 females and eggs of Meloidogyne salasi sp. n. from rice, cv. C.R.1113 41 8 Measurements of 50 males of Meloidogyne salasi sp. n. from rice, cv. C.R.1113 55 9 Measurements of 50 second-stage juveniles of Meloidogyne salasi sp. n. from rice, cv. C.R.1113 63 0 Response of seven differential plants to three populations of Meloidogyne incognita from Florida 81 1 Interpretation of the predominant type of perineal pattern of females of three host races of Meloidogyne incognita from Florida 82 2 Comparative morphological data (pm) from females of three host races of Meloidogyne incognita from Florida 85 3 Comparative morphological data (pm) from second-stage juveniles of three host races of Meloidogyne incognita from Florida 36 4 Comparative morphological data (pm) from males of three host races of Meloidogyne incognita from Florida 87 v

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Table Page 15 Range of measurements of females from 16 populations of Meloidogyne spp. from Costa Rica 108 16 Range of measurements of males from 16 populations of Meloidogyne spp. from Costa Rica 109 17 Range of measurements of infective second-stage juveniles from 16 populations of Meloidogyne spp. from Costa Rica.. 110 18 Range of measurements of females from three host races of Meloidogyne incognita from Florida Ill 19 Range of measurements of second-stage juveniles from three host races of Meloidogyne incognita from Florida 112 20 Range of measurements of males from three host races of Meloidogyne incognita from Florida 113 vi

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LIST OF FIGURES Fi S ure Page 1 Map of Costa Rica showing the approximate location of the collection sites of 16 populations of Meloidogyne spp 12 2 Photomicrographs of female perineal patterns of nine populations of Meloidogyne spp. from Costa Rica. CR4: M. arenaria . CR1, 3, 5, 6, 11, 12, 16, and 17: M. incognita 20 3 Photomicrographs of female perineal patterns of six populations of Meloidogyne spp. from Costa Rica. CR2: M. sp.. CR7, 9: M. exigua. CR10, 14, and 15: M. hap la 21 4 Outlines of females of Meloidogyne salasi sp. n 43 5 Female of Meloidogyne salasi sp. n 44 6 Scanning electron photomicrographs of face views of females of Meloidogyne salasi sp. n 46 7 Cephalic region of a female of Meloidogyne salasi sp. n. . 48 8 Anterior region of a female Meloidogyne salasi sp. n 49 9 Photomicrographs of female perineal patterns of Meloidogyne salasl S P« n.. A, B and C from Costa Rica. D from Panama. 51 10 Perineal patterns of Meloidogyne salasi sp. n.. A, B and C from Costa Rica. D from Panama 52 11 Scanning electron photomicrographs of female perineal pattern of Meloidogyne salasi sp. n 53 12 Males of Meloidogyne salasi sp. n.. A Esophageal region (ventral). B Cephalic region (lateral). C,D Tail (lateral) ^ 13 Anterior region of males of Meloidogyne salasi sp. n. . A,B Scanning electron microscope photomicrographs. C,D Light microscope photomicrographs 53 14 Scanning electron photomicrographs of males of Meloidogyne salasi sp. n. . A,B Face views. C Lateral fields — D Tip of spicules showing pores 60 vii

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Figure Page 15 Second-stage juveniles of Meloidogyne salasi sp. n. . A Esophageal region (lateral) . B Cephalic region (lateral). C Tail (dorsal). D Tail (lateral) 64 16 Scanning electron photomicrographs of face views of secondstage juveniles of Meloidogyne salasi sp. n 65 17 Lateral field of second-stage juvenile of Meloidogyne salasi sp. n 777; 66 18 Second-stage juveniles of Meloidogyne salasi sp. n.. A,B Anterior region. C,D Tail terminus 68 19 Photomicrographs of female perineal patterns of three populations of Meloidogyne incognita from Florida 83 Diagramatic sketch of comparative electrophoretic patterns of some Meloidogyne spp. from Costa Rica and Florida. Left to right: M. salasi sp. n.; M. exigua (CR7) ; M. exigua (CR9) ; M. hap la (CR10); M. hapla (CR14); M. arenaria ; M. incognita (CR3) ; M. incognita (CR11); M. incognita (CR12); M. incognita (CR16) ; M. incognita (M-195); M. incognita (M-165) and M. incognita (M-198) 98 viii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DIFFERENTIAL PLANT RESPONSES, MORPHOMETRICS AND ELECTROPHORETIC PATTERNS OF SOME Meloidogyne spp. FROM COSTA RICA AND FLORIDA, U.S.A., AND THE DESCRIPTION OF Meloidogyne salasi sp. n. By Roger Lopez Chaves August 1984 Chairman: D.W. Dickson Major Department: Entomology and Nematology Based on a morphometric study of males, females and second-stage juveniles, and on the responses of seven differential plants, five species of Meloidogyne were distinguished among 16 populations collected at different locations in Costa Rica. These were M. arenaria , M. incognita , M. hapla , M. exigua and an undescribed species, found infecting rice. The responses of the differential plants indicated that the M. arenaria population was host race 2 (does not infect peanut) and that the M« incognita populations included host races 1 and 2. Evidence of pathogenic variation was found between two M. exigua populations. One reproduced readily on tomato, whereas the second population did not. Similarly, two populations of M. hapla reproduced readily on pepper, whereas the third population reproduced only to a limited extent on that host. Three populations of M. incognita from Florida, U.S.A., were distinguished as host races 1, 2, and 3 based on morphometries and ix

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differential plant responses. These were compared to ten populations of Meloidogyne spp. from Costa Rica by means of starch gel electrophoresis. With a few exceptions, malate dehydrogenase, phosphoglucose isomerase, fumerase, a-glycerophosphate dehydrogenase, and isocitrate dehydrogenase isozyme patterns could be used to differentiate the species of Meloidogyne that were investigated. Intraspecif ic differences were also noted in patterns of the five enzymes between the two populations of M. hap la and in the patterns of all enzymes except isocitrate dehyrogenase between the two populations of M. exigua . Meloidogyne salasi sp. n. , a pathogen of rice ( Oryza sativa L.) in Costa Rica and Panama, was described and illustrated. It can be distinguished from related species (M. kralli , M. acronea and M. graminis ) by the areolation of the lateral fields in the male, the dimensions and characters of the perineal pattern of the females, and by the total length and a, b, and tail length/anal width ratios of the infective second-stage juveniles. x

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CHAPTER I INTRODUCTION The broad geographical distribution, wide host range, severe pathogenic effects and synergistic interactions with many other kinds of plant disease organisms, have placed root-knot nematodes ( Meloidogyne Goeldi, 1887, Nematoda: Meloidogynidae) among the major plant pathogens affecting man's food supply (Taylor and Sasser, 1978; Sasser, 1980; Sasser and Carter, 1982). Practically every crop grown is infected by one or more species of this genus. Not only are yields affected but quality is also reduced, particularly in the case of root crops. Management strategies aimed at reducing the severity of the damage caused by Meloidogyne spp. include the use of chemicals, crop rotation, resistant cultivars and other cultural practices (Taylor and Sasser, 1978; Sasser and Carter, 1982). The last three tactics require extensive knowledge of the morphology, variability and ecology of the species causing the damage. One of the problems associated with the implementation of nonchemical management tactics against root-knot nematodes is the correct identification of populations. Identification is complicated by the variation in morphology and host range commonly present in species of this genus (Netscher, 1978; Whitehead, 1968). Due to this variability, approaches other than classical morphology have been used to identify species. Among those are differential host test (differential plants) 1

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(Taylor and Sasser, 1978), biochemical analysis of different enzymatic and nonenzymatic proteins (Hussey, 1982), cytology, and mode of reproduction (Triantaphyllou, 1982). The identification and/or quantification of the variability within and among species of root-knot nematodes by these different approaches could provide the basis for a better understanding of the genus, not only from the morphological, but from the physiological and ecological points of view as well. This understanding would enable recognition of those characters which are species specific and therefore reliable for distinguishing species, as well as recognition of characters with little or no value in the identification of field populations due to their overlap among or between species or their instability. Eventually, this could lead to the development of a faster, more accurate methodology for the identification of species and/or races within a species. We would then have a better basis for the planning and implementation, both locally and internationally, of management strategies for this important group of plant pathogens. Research was initiated in late 1980 with four objectives: a) to study the variability of some populations of root-knot nematodes from Costa Rica by a morphometric characterization of males, females, and infective second-stage juveniles, and by their reaction on certain differential plants; b) to characterize three populations of M. incognita (Kofoid and White, 1919) Chitwood, 1949 from Florida, U.S.A., by morphometries and the responses of differential plants, and to compare them to populations of this same species from Costa Rica; c) to use starch gel electrophoresis to differentiate several species of root-knot nematodes

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3 found in Costa Rica, compare populations of M. incognita from both Costa Rica and Florida, and investigate possible differences among the latter; and d) to describe and illustrate a new species of root-knot nematode found infecting rice in Costa Rica and Panama.

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CHAPTER II VARIABILITY OF Meloidogyne spp FROM COSTA RICA Introduction Papilla and Yuquilla, meaning small potato and small cassava, are common names applied by some farmers in Costa Rica to the root-knot disease of numerous cultivated plants. Others simply call the disease Nematodos or Meloidogyne . Until recently, the reports of root-knot nematodes in Costa Rica were scattered, the first one apparently being that of von Bulow (1934). This author reported "vermes of the genus Heterodera " inside abnormal soybean nodules and in galled peach roots. A year later Heterodera was again found on peaches in San Pedro de Montes de Oca (von Bulow, 1936) . By 1935 root-knot nematodes were found in melon, cabbage and tobacco, in addition to coffee and Inga (von Bulow, 1935). Some observations were published on the occurrence of nematodes on Inga and coffee collected from several locations in the Central Plateau (von Bulow, 1937) . Many of the die-back problems in coffee were attributed to the infection by nematodes. His illustrations of root galls on both plant species, as well as those of nematode eggs, juveniles and females, seemed to correspond to a species of the genus Meloidogyne , as pointed out previously by Salas and Echandi (1961). Sixteen years later M. incognita var. acrita Chitwood, 1949, was identified for the first time in Costa Rica (Taylor and Loegering, 1953). These authors reported a low incidence of this species in abaca. 4

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Olsen and Thomas (1954) successfully controlled M. incognita var. acrita on tomato and okra with DD and EDB. They also found aldrin and parathion did not give satisfactory results. A few years later Van der Laat (1960) successfully controlled M. incognita on tomato with DBCP, 1,3-D and DD in a sandy loam soil but not in a clay loam. More recently, Ramirez (1971) did not obtain yield increases in tomato with the application of ethoprop at 10 and 20 kg ai/ha, but there were reductions in the number of nematodes in the soil and in the rootknot index up to three months after transplanting. He found that fensulfothion gave higher yields than ethoprop at the same rates, although its effect on M. incognita was not as severe as that of ethoprop. Salas and Echandi (1961) demonstrated the pathogenicity of M. exigua Goeldi, 1887, on coffee seedlings. They mentioned that under field conditions plants infected by this nematode showed above ground symptoms of wilting, chlorosis, defoliation and low yields, whereas below ground galls appeared mostly on the finer roots. They considered this nematode induced a serious disease of coffee. In 1968 Figueroa (1973) found a species of root-knot nematode infecting rice in Volcan de Buenos Aires, Puntarenas. He later studied its life cycle and illustrated some of its morphological characters. He also demonstrated its pathogenicity on 12 rice genotypes. Salas (1975) mentioned the presence of M. incognita in the Atlantic zone, the Central Plateau and the high mountains of Costa Rica. He also reported that M. exigua , M. hap la Chitwood, 1949, and M. javanica (Treub, 1885) Chitwood, 1949, were common in the Central Plateau.

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6 Another report about Meloidogyne in Costa Rica is that of Pessoa (1973). He found root-knot nematode juveniles in virgin soil in the Atlantic zone that was to be planted with bananas. Recent surveys (Gonzalez, 1978a; Gonzalez, 1978b; Gonzalez, 1979; Lopez, 1978; Lopez, 1980c; Lopez and Azofeifa, 1980; Lopez and Azofeifa, 1981; Lopez and Salazar, 1978; Lopez et al., 1980) and collections of field populations (Alvarado and Lopez, 1982; Hidalgo and Lopez, 1980a; Salazar, 1980a; Salazar and Lopez, 1980) further showed the wide host range and widespread occurrence of root-knot nematodes throughout the country. The species mentioned were M. hapla , M. exigua and M. javanica . An undescribed species was also found infecting rice in the southeastern part of the country (Alvarado and Lopez, 1981; Lopez, 1981a; Sancho, 1981) . Numerous crops and weeds were cited as hosts of the different species of root-knot nematodes, including some new ones (Lopez, 1980c; Lopez and Salazar, 1978). The pathogenicity of these species of root-knot nematodes on several crops is beginning to be studied. Results of experiments carried out with M. incognita on lettuce (Castro and Lopez, 1981; Gonzalez and Lopez, 1980b), common bean (Lopez, 1980b), corn (Hidalgo and Lopez, 1980b) and with the undescribed species on rice (Sancho, 1981) showed that rootknot nematodes can significantly reduce the growth and/or yield of these crops. These losses can be even higher if the nematodes interact with other plant pathogens, as was shown with Fusarium oxysporum f . sp. pisi and M. incognita plus M. hapla on green peas (Padilla et al., 1980). Indirect evidence of the damage root-knot nematodes can inflict on both field and vegetable crops was obtained with the experimental use of nematicides and other management practices. In air-cured tobacco, the

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7 application of DD increased yield by 20% (Lopez and Fonseca, 1978), whereas materials such as ethoprop and carbofuran increased yields of burley and flue-cured tobacco by 9 and 10%, respectively (Calvo and Lopez, 1980; Carrillo and Lopez, 1979). Greater increases were obtained in certain vegetable crops. For instance, metham-sodium, phenamiphos , carbofuran and aldicarb increased carrot yields 238, 135, 114, and 72%, respectively over the untreated controls (Perlaza et al., 1979). In lettuce, the same chemicals increased yields by 112, 219, 117, and 91%, respectively over the control (Perlaza et al. , 1978). Both of these experiments were carried out under high initial population densities. However, when the initial population density was relatively low for lettuce, the increase was only 13% with phenamiphos (Mattey and Lopez, 1978). Some nonfumigant nematicides have also given satisfactory results on green peas (Padilla and Lopez, 1979). Yield increases of 35, 21, 18, and 27% were obtained with aldicarb, two commercial formulations of carbofuran and with ethoprop, respectively. Use of phenamiphos or fensulfothion significantly reduced the root-knot index and the density of juveniles in the soil at harvest time, but failed to increase the yield. It appeared that these two chemicals may be phytotoxic to green peas. In celery, the elimination of the previous crop residue (roots) severely infected with M. incognita increased yields by only 3%. The incorporation of organic matter (a mixture of broiler manure and sawdust) caused a 13% decrease in celery weight and promoted significantly higher root-knot indices 46 and 94 days after transplanting. The application of aldicarb did not affect celery yield (Incer and Lopez, 1979). In a

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8 subsequent study (Rivera and Lopez, 1982), carbofuran, phenamiphos and aldicarb increased the yield of the celery cultivar 'Dwarf, but these materials failed to produce higher yields with the cultivar '5205'. Ethoprop and phenamiphos apparently had a phytotoxic effect. In corn, no significant differences in nematode populations at harvest time or yield were found between carbofuran, fensulf othion, phenamiphos and ethoprop and the untreated control (Gonzalez and Lopez, 1980a). A few morphological and morphometric studies of some populations of root-knot nematodes were conducted in the recent past (Hidalgo and Lopez, 1980a; Lopez and Salazar, 1978; Salazar, 1980a; Salazar and Lopez, 1980). M. hapla was found widespread in the Central Volcanic Range, at altitudes between 1,360 and 2,501 m above sea level. This area has average temperatures between 14 and 18°C, with precipitation over 2,000 mm per year. The soil type is Andept. In most cases the range and average values of several characters of second stage juveniles, and of certain characters of the perineal pattern, were similar to those reported previously for this species, although some discrepancies were found. For instance, the striae of the perineal pattern were mostly unbroken, in contrast with the report by Esser et al. (1976), and a dilated rectum was observed in some second-stage juveniles. In both this and another study (Salazar and Lopez, 1980) with second stage juveniles of M. hapla , significant differences among populations were found in the distance between the dorsal esophageal gland orifice and the base of the stylet knobs (DEGO) , tail length, maximum body width, anal width and the a ratio. Differences among populations were also

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9 noted in the c ratio (Salazar and Lopez, 1980) and in the body length (Lopez and Salazar, 1978). A similar situation was found for M. incognita ; i.e., the range and mean values for females, males and second-stage juveniles were similar to those reported earlier for this species, but some significant differences for certain characters were found among populations (Hidalgo and Lopez, 1980a; Salazar and Lopez, 1980). This species is the most prevalent one in agricultural fields (Alvarado and Lopez, 1982; Hidalgo and Lopez, 1980a; Lopez and Azofeifa, 1981), and its dissemination does not seem to be associated with any particular set of environmental conditions Differences among three populations of M. javanica in total length, base of stylet to head end, DEGO, tail length, maximum body width, anal width and a ratio of second-stage juveniles, stylet, maximum body width, DEGO and spicules (measured as the chord of their arch as described by Chitwood (1949)) of males, and stylet and DEGO of females were reported also (Salazar, 1980a). The differential host test (Taylor and Sasser, 1978) was performed also on a few populations of root-knot nematodes (Salazar, 1980a; Salazar and Lopez, 1980). In general, the reaction of the hosts could be considered "typical" for each of the species tested, except in the case of M. javanica , where one population was able to infect 'California Wonder' pepper (Salazar, 1980a), and the failure of several populations of M. hap la to reproduce on 'Tioga' strawberry (Salazar and Lopez, 1980). Race 1 of M. incognita was the only one found by Salazar and Lopez (1980). The spatial distribution of Meloidogyne spp. under field conditions is another aspect that was studied in some detail. The densities of

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10 M. incognita (Gonzalez, 1978a) and of a mixture of M. incognita and M. hapla (Perlaza et al., 1978; Perlaza et al., 1979) in vegetable fields varied greatly even in adjacent small plots. Similar observations were performed on rice regarding an undescribed species of Meloidogyne (Lopez, 1981a). This nematode was also concentrated in the upper 15 cm soil layer, and its density decreased sharply as sampling depth increased. In bur ley tobacco, densities of second-stage juveniles of M. incognita were greater in the horizontal plane 5-10 cm away from the trunk, and in the upper 15 cm soil layer. Densities decreased as sampling depth increased, but not to a large degree. These data were taken one week after harvest (Lopez, 1981b). In sugarcane, higher densities of Meloidogyne spp. were located 15 cm away from the plants in the horizontal plane; vertically, the highest densities were located between 61 and 75 cm deep (Salazar, 1980b). The influence of soil type and extraction method on the recovery of Meloidogyne spp. was studied. Significantly higher densities of M. incognita juveniles were extracted from an Ustropept soil when it was washed in water three times. Higher numbers were recovered from Ustult and Distropept soils when they were suspended 20 and 60 seconds in water, respectively, before being poured through the sieves. An arrangement of one 50-mesh sieve nested on top of two 325-mesh sieves and a 1.12 sp. g. sugar solution recovered higher densities from the Ustropept and the Distropept soils, respectively. Significantly more juveniles were extracted from the three soil types with the centrifugal-flotation technique than with the modified Baermann funnel (Alvarado and Lopez, 1982).

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11 Different variations of both the centrifugal-flotation and the modified Baermann funnel did not improve the extraction of juveniles of an undescribed species of root-knot nematode from a rice field. More juveniles were recovered with the centrifugal-flotation method (Alvarado and Lopez, 1981) . Another interesting observation on root-knot nematodes was that in four females of M. incognita some eggs developed to second-stage juveniles while they remained in the uterus (Perlaza and Lopez, 1979). Materials and Methods Nematode Populations Sixteen populations of root-knot nematodes were collected from different localities in Costa Rica (Fig. 1) and increased in a greenhouse at the Facultad de Agronomla, Universidad de Costa Rica, San Pedro. Some selected ecological characteristics of the collection sites of these populations, along with the hosts on which they were collected and their population designation, are presented in Table 1. The inoculum for the propagation of each population consisted of several dozen egg masses collected from roots of the host from the original locality where the population was collected. Most populations were increased on tomato, cv. Rutgers. Populations CR7 and 9 were maintained on coffee, cv. Caturra, whereas population CR2 was maintained on rice, cv. C.R. 1113. All plants were grown in 2,000 cm 3 clay pots that contained 1,700 cm 3 of an Andept soil (43.2:31.4:25.4% sand: silt: clay; 8.7% O.M. and 5.8 pH) . The soil in all cases was treated with steam at 105°C for 24 hours prior to use. Each pot was fertilized twice a week during the first five weeks of plant growth with 150 ml of a 1% 20-20-20 fertilizer formula solution. Air temperatures varied between 17 and 31°C.

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12 840 0' S4 00' 1. Map of Costa Rica showing the approximate location of the collection sites of 16 populations of Meloidogyne spp..

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13 ca o •H oc o iH o u w id o o c o cd co > • cu cd 4J co 0 a a. o 4-1 CO 4-1 4-1 4-1 4-1 4J 4-1 0) CO CO 03 CO CO CO u 01 01 n u CO o CO CO o CO u [s 0 0 •H 4J 4J 4H 4J •H •H 4-1 4-1 4-1 •H 4-1 4-1 0 CU 4J CO CO O o 01 o cu cu a e 3 rH •H 4-1 •H 9 § c E c c d 4-1 4-1 CU o O •H cd cd cd cu 0) cu CU -O 1 a 0) 1) 0 cd cu 0) 4-1 4J 4J * c c c C c h c B c c a cd cd H H r-H rH cd cd cd cd cd o o 0 H rH 4-1 4-1 Cd cd cd cd 4J 4-1 4J cu 4-1 4-1 S E E cd crj c c co CJ u u B c c c c a a u o o cd •H •H •H o o o cd o c 1-1 U "H 6 6 xi a ex o. 6 § S 4-1 E E cu a. a 0) cu 0 c o o> 1) 0) c CU B i 1 o o M M M 1-1 rH r< >n o >H u 8 In M Cm P-i H H H Cm Ph Ph sc =p* J H H cd E cd «H 4-1 O CO cd cd co cd N rl o rH H 01 Z cd 4J TJ C cd a 3 cu os u o O CN O in H* CN m CN cd c ca < -H 3 -H Cd rJ 4-i a G B u cd cd cd CO CO CO moo O vO CN On CO O C O •H CO ri > H a o o o o o VO rH vo CN 00 rH 00 CN CO CO CM -J lO Oi sr oo oo vO \T\ —i rH CN rH VO 00. 00 00 00 CO CO H H (O i-H CN CO m cm m CO ~3" r-» r-~ t-~ oo oo oo oo r» co o\ o o CO VO rH rH CN CO vO CN sr 4* CO CO CM -H 00 rH CO oo m o vo m co m sr cm CO sf CO oo oo cn vo m in cm co CO CO U C ^4 aj 0 t4 S m z 4-1 r-l o CO cd CO CO •H CO Sh u co O. (H cd CO 0) o E •H M E a u Cd E C i— i a" Sh iH o 11 CO CO O rH Cd •H •H Cm Ph Pm 0PS Ph m o r-» rH 00 rH m vo vo vo in oo o O oo CO rH CM CO rH o o o 00 CO co oo m -dCO CN O o vO r1 • • o in CM co 00 m i — i CN co os PC PS u CJ> U hjm vo PS OS DS UOCJ o N » H os os os u a c_> OS O os CO sr m VO rH i — i —i rH — i OS os OS PS OS U u U c_>

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14 Morphology Twenty specimens were used for each character studied in the males, females and infective second-stage juveniles. All measurements were analyzed statistically with a one-way classification model, and the mean values were compared using the Duncan's Multiple Range Test. The perineal patterns were prepared according to the method described by Franklin (1962) and later modified by Taylor and Netscher (1974), but without staining the roots. Each perineal pattern was divided into several zones, following the method described by Esser et al. (1976). To obtain infective second-stage juveniles, eggs from several egg masses collected at random from the host roots were kept in a small petri dish for 24 hours. Live infective second-stage juveniles were placed on glass slides in a drop of distilled water, ringed with Zut® and a coverslip applied. Fifteen to 20 minutes after mounting, the juveniles were observed with an ordinary compound microscope (LM) . Juveniles lying in a straight plane were measured and data recorded. Males were obtained by dissecting galled roots in tap water. The males were mounted using the same method as described for the juveniles. Occasionally, the slide was maintained for 24 hours to facilitate the observation of the lateral fields. All measurements were made with a calibrated ocular micrometer at 1.500X magnification (oil immersion objective), except for the female length and maximum body width, and the second-stage juvenile length, which were measured at 150X.

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15 Differential Plants The reactions of seven differential plants (Taylor and Sasser, 1978) were evaluated for each of the 16 populations of root-knot nematodes. Plants used were tomato, cv. Rutgers; tobacco ( Nicotiana tabacum L.), cv. NC-95; pepper ( Capsicum annum L.), cv. California Wonder; corn ( Zea mays L.) , cv. Minnesota A-401; cotton ( Gossypium hirsutum L.), cv. Deltapine 16; peanut ( Arachis hypogea L.), cv. Florunner, and watermelon ( Citrullus vulgaris Schard) , cv. Charleston Grey. Seeds of the plants, except tobacco, were germinated over filter paper in petri dishes at 28°C for three days; the germinated seeds were then placed in small plastic pots containing steam-pasteurized soil. Seedling ages at inoculation were as follows: corn — 10 days old, peanut, cotton and watermelon — 20 days old, tomato — 30 days old, and tobacco and pepper — 60 days old. Inocula of all populations, except CR2, 7 and 9, were obtained from a galled root system of tomato, which was cut into small pieces and treated with a 1% sodium hypochlorite solution (Hussey and Barker, 1973). Coffee roots infected with populations CR7 and 9 were cut into small pieces and then chopped in a blender for 20 seconds; the resulting material was treated with the 1% sodium hypochlorite solution. The galled rice roots used to collect the inoculum of CR2 were macerated in a blender for 15 seconds and then washed with tap water on a 200-mesh sieve nested on top a a 500-mesh sieve; a strong jet of water was applied and the eggs were recovered from the 500-mesh sieve. About 10,000 eggs were pipetted over 1,000 cm 3 of soil, the seedlings transplanted and 700 cm 3 of soil added. Pots were placed randomly on the greenhouse bench, and each population was isolated by plastic

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16 dividers to avoid contamination. Each host-population combination was replicated four times. Fifty-five to 60 days after inoculation the plant roots were washed free of soil and immersed for 30 minutes in a 0.0016% Phloxine B solution (Dickson and Struble, 1965) to stain the egg masses. Each root system was rated according to the number of egg masses, using the following scale (Taylor and Sasser, 1978): 0: 0 egg masses 3: 11-30 egg masses 1: 1-2 egg masses 4: 31-100 egg masses 2: 3-10 egg masses 5: more than 100 egg masses. In certain cases, perineal patterns were prepared from females on certain differential plants to insure that the species recovered was the same used for the inoculation. Results Five species of Meloidogyne were identified among the 16 populations studied. These species were M. incognita (populations CR1, 3, 5, 6, 11, 12, 16, and 17), M. exigua (populations CR7 and 9), M. hapla (populations CR10, 14, and 15), M. arenaria (population CR4) , and an undescribed species (population CR2). Population CR13 was identified as a mixture of M. incognita and M. hapla . Morphology The interpretation of the predominant type of perineal pattern for each species is presented in Table 2. Only specimens of the undescribed species (CR2) and a few from M. exigua (CR7 and 9) had a posterior protuberance. Populations CR16 and 17 of M. incognita had a few striae originating at the vulval lips and going out to the sides. M. exigua (CR7 and 9) had three striae in the perineum, whereas M. hapla (CR10,

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17 Table 2. Interpretation of the predominant type of perineal pattern of females of 16 populations of Meloidogyne spp. from Costa Rica. Vulva Pop. Posterior lip Perineum Lateral Striae zone protuberance striae striae incisures 1 9 "5 -> A M. incognita A A A A F FWB FWB FWR CR3 A A A A F FWB CR5 A A A A F FWB i WD CR6 A A A A F FWB FWB FWB CR11 A A A A F FWB FWB FWB /IT) 1 O CR12 A A A A F mi>tr 171WD MTJT? PIWd CR16 A F A A M MWB MWB MWB CR17 A F A A F FWB FWB FWB M. exigua CR7 A A 3 I F FSB FSB FSB CR9 A A 3 I F F9R r jd rjJl M. hapla CR10 A A 1 I F FSU FSU FSU CR1A A A 1 I F FSU FSU FSU CR15 A A 1 I F FSU FSU FSU M. arenaria CR4 A A A I F FSB FSB FSB M. sp. CR2 P A A A F FSU FSU FSU M. incognita & M. hapla CR13 A A 1 & A 1 & A F FSW& FSU& FSU& FWB FWB FWB A: absent; P: present; F: few; M: moderate in number; W: wavy; B: broken; U: unbroken; S: smooth; I: interrupted.

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18 14, and 15) and some specimens of CR13, a population consisting of a mixture of M. incognita and M. hapla , had one stria. The striae of M. arenaria (CR4), M. exigua (CR7, 9) and M. hapla (CR10, 14, 15) were interrupted where the lateral lines normally are, but they were not distinct enough to be considered lateral lines. The M. incognita populations CR1, 3, 5, 6, 11, and 17 had a few, wavy and broken striae in zones 2, 3, and 4, whereas CR12 and 16 had a moderate number of striae in these zones. In these same zones M. exigua (CR7, 9) had few, smooth, broken striae, whereas M. hapla (CR10, 14, 15) had few, smooth, unbroken striae. M. arenaria (CR4) had few, smooth, broken striae in zones 2, 3, and 4. In the mixture of M. incognita and M. hapla (CR13) perineal patterns with few, smooth and unbroken striae, as well as perineals with few, wavy, broken striae in zones 2, 3, and 4 were found. The undescribed species (CR2) had few, smooth, mostly unbroken striae in zones 2, 3, and 4. Striae of the undescribed species and of M. hapla were relatively fine whereas they were relatively coarse in the other species. The shape of the perineal pattern varied with the species. In M. incognita the perineal patterns of all populations were mostly pyriform, with a trapezoidal dorsal arch. The perineal patterns of the two M. exigua populations were roughly rounded, with a low rounded dorsal arch; the striae were rather coarse and far apart. In M. hapla populations the perineal patterns were roughly rounded, with a low and wide dorsal arch. No wings were observed in the perineal patterns, but punctations on the tail terminus area were present. The striae were closely spaced. In M. arenaria the perineal patterns were mostly oval, with striae forming a shoulder on the low, flat to rounded dorsal arch. In the mixture

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19 of M. incognita and M. hapla both pyriform and roughly rounded perineal patterns were found. The undescribed species (CR2) had oval shaped perineals, with high and wide rectangular dorsal arches; the striae were far apart. A photomicrograph of one perineal pattern from each population, except of the mixture of M. incognita and M. hapla , is presented in Figs. 2 and 3. The mean values of morphometric characters of the females are presented in Table 3. Highly significant differences among populations were found in stylet, DEGO, distance between the middle of the excretory pore and the head end (excretory pore), maximum body width, body length, vulva, anus-vulva and interphasmidial distances. The ranges for these characters are presented in Table 15 in the appendix. In general, the ranges of all characters overlapped to some degree among populations. Average values for the characters measured in infective secondstage juveniles are presented in Table 4, and their ranges are presented in Table 16 in the appendix. Highly significant differences among populations were found in total length, tail length, maximum body width, anal width, stylet base to head end, DEGO, and the a and c ratios. Undilated recta were present in M. exigua (CR7, 9) and M. hapla (CR10, 14, 15), whereas they were dilated in the other populations. In all juveniles the hemizonid was located anterior to the excretory pore. The range of total length of M. exigua (CR7, 9) did not overlap with that of the undescribed species (CR2) . A similar situation was found between the M. hapla populations CR14 and 15 when compared to the undescribed species (CR2) . Range values for the M. hapla population CR10 did overlap with that of the undescribed species, although it did not with the range values of the other two populations of M. hapla (CR14, 15). There

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20 Fig. 2. Photomicrographs of female perineal patterns of nine populations of Meloidogyne spp. from Costa Rica. CR4: M. arenaria . CR1, 3, 5, 6, 11, 12, 16, and 17: M. incognita .

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21 Fig. 3. Photomicrographs of female perineal patterns of six populations of Meloidogyne spp. from Costa Rica. CR2: M. sp.. CR7, 9: M. exigua . CR10, 14, and 15: M. hapla .

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22 Table 3. Morphometric characters of females of 16 populations of Meloidogyne spp. from Costa Rica. Exc. pore Stylet M. incognita CR1 23.7 a 15.2 cde 4.1 be 486 efg 634 bed CR3 ZO . 4 abed i / t abed 4. 0 abc 538 g 764 gh CR5 26.2 abed 15.9 e 3.8 ab 459 def 679 c ds f CR6 23.4 a 15.6 de 4.0 abc 519 g 721 fg CR11 abc it t; 1 J . j ab 3.9 ab 495 efg 784 h CR12 26.4 bed 15.4 cde 3.4 a 439 de 656 bede CR16 31.7 cde 15.4 cde 4.6 cd 371 be 642 bede fRI 7 9/i 7 ab i e o 1 5. z cde 4.3 bed 471 def 659 bede M. exigua CR7 35.3 e 14.8 bede 6.1 e 272 a 493 a CR9 bede U A bed 4.0 e 325 ab 491 a ri . nap j. a CR10 34.8 e 12.9 a 5.7 e 472 def 697 ef CR14 36.5 e 13.7 ab 5.7 e 423 cd 629 be CR15 33.3 de 14.2 abc 5.8 e 486 efg • 728 fg M. arenaria CR4 34.2 e 15.5 cde 4.6 d 508 fg 698 ef M. sp. CR2 35.4 e 13.6 ab 3.9 ab 468 def 602 b M. incognita & M. hapla CR13 25.2 abc 15.6 de 4.2 be 495 efg 690 def CV (%) 28.4 10.1 14.6 14.6 9.9 DEGO refers to the distance between the base of the stylet knobs and the dorsal esophageal gland orifice. * Mean of 20 observations. All measurements in ym. Means in the same column followed by the same letter do not differ significantly from one another according to Duncan's Multiple Range Test (P = 0.01).

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23 Table 3-continued. Pop. Vulva length Anus-vulva Interphasmidial distance CR1 22.1 bcde 17.8 cde 24.5 def CR3 25.3 f 18.9 ef 28.6 h CR5 f i 10 . 0 del 27.2 CR6 24.5 ef 17.0 abede 27.5 gh CR11 23.2 bcdef 19.8 f 26.2 fg CR12 abed 1 / . D abede 23 .0 bed CR16 23.5 def 18.1 cdef 26.6 g CR17 23.3 cdef 18.0 cde 26.9 gh M. exigua CR7 18.9 a 17.2 abede 24.8 ef CR9 abed 1 A 7 a bed oo c be M. hapla CR10 20.7 abed 15.7 ab 21.7 b CR14 20.8 abed 15.6 a 24.9 ef CR15 20.4 abc 17.6 bcde 22.8 bed M. arenaria CR4 21.5 abed 17.8 cde 27.6 gh M. sp. CR2 23.2 bcde 16.3 abc 15.1 a M. incognita & M. hapla CR13 20.3 ab 17.5 abede 23.9 cde CV (%) 14.2 11.7 7.9 DEGO refers to the distance between the base of the stylet knobs and the dorsal esophageal gland orifice. ** Mean of 20 observations. All measurements in ym. Means in the same column followed by the same letter do not differ significantly from one another according to Duncan's Multiple Range Test (P = 0.01).

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24 Table 4. Morphometric characters of second-stage juveniles of 16 populations of Meloidogyne spp. from Costa Rica. Total Tail Maximum Pop. length length body width Anal width M. incognita CR1 414 cd 53.3 b 14.9 abc 10.8 cde CR3 c /i Q Q a 1 C 1 1 o . 1 e 11 1 J 11.1 de CR5 426 d 53.9 be 15.2 bed 10.8 cde CR6 449 e 57.5 d 14.9 abc 10.9 cde CR11 £90 j a JZ . 0 D 1 C 1 1-) . 1 bed 11 1 J 11.1 de CR12 419 d 49.8 a 15.6 cde 10.3 bede CR16 461 e 56.4 cd 14.9 abc 9.0 a CR 1 7 •jo/; JoD D H 1 . 4a 15.7 de 10.3 bede M. exigua CR7 373 ab 48.5 a 14.5 ab 9.4 ab CR9 368 a 48.8 a 14.7 ab 9.4 ab M. hapla CR10 464 f 61.2 e 14.7 ab 10.6 bede CR14 373 ab 47.4 a 14.6 ab 10.2 bed CR15 373 ab 48.1 a 14.5 ab 10.0 abed M. arenaria CR4 459 e 57.3 d 14.2 a 9.8 abc M. sp. CR2 466 f 69.5 f 16.1 e 11.4 e M. incognita & M. hapla CR13 418 d 54.7 bed 14.9 abc 10.3 bede CV (%) 4.0 6.3 5.5 12.0 DEG0 refers to the distance between the base of the stylet knobs and the dorsal esophageal gland orifice. ** Mean of 20 observations. All measurements in um. Means in the same column followed by the same letter do not differ significantly from one another according to Duncan's Multiple Range Test (P = 0.01). All juveniles had the hemizonid anterior to the excretory pore. In CR7, 9, 10, 14, and 15 the recta were undilated, whereas in the remaining populations they were dilated.

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25 Table 4-continued . Stylet base Pop. to head end DEGO M. Incognita CR1 15.3 de 3.2 ab 27.9 bed 7.8 bede CR3 15.6 ef 3.1 ab 26.2 abc 8.1 ef CR5 1 S 7 of J . u a O Q O 2 o . j bed 7.9 cdef CR6 15.5 ef 3.1 ab 30.3 def 7.8 bede CR11 15.1 cd 3.1 ab 28.0 bed 8.0 def CR12 IS L J. J • H Ucl J . 0 j Q 23.5 a 8.4 g CR16 15.8 f 3.6 cd 31.2 def 8.2 fg CR17 15.5 ef 3.2 ab 25.0 ab 8.2 fg M. exigua CR7 13.6 a 3.2 ab 25.6 ab 7.7 bed CR9 a j.j Dcd 2 j . 1 ab T c 1_ 7.5 b 11* LldUld CRIO 14.8 c 4.5 e 31.7 ef 7.6 be CR14 14.2 b 4.2 e 25.7 ab 7.9 cdef CR15 14.0 b 3.7 d 25.8 ab 7.8 bede M. arenaria CR4 15.4 def 3.5 bed 32.6 f 8.0 def M. sp. CR2 14.2 b 3.3 abc 29.2 cde 6.7 a M. incognita & M. hapla CR13 15.3 de 3.1 ab 28.2 bed 7.6 be CV (%) 3.2 12.7 13.5 4.7 DEGO refers to the distance between the base of the stylet knobs and the dorsal esophageal gland orifice. * Mean of 20 observations. All measurements in urn. Means in the same column followed by the same letter do not differ significantly from one another according to Duncan's Multiple Range Test (P = 0.01). All juveniles had the hemizonid anterior to the excretory pore. In CR7, 9, 10, 14, and 15 the recta were undilated, whereas in the remaining populations they were dilated.

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26 was no overlap in the range of total length either between M. arenaria (CR4) and M. exigua (CR7 , 9), or between M. arenaria (CR4) and the M. hapla population CR15. Tail length ranges did not overlap when the undescribed species was compared to M. exigua (CR7, 9), two M. hapla populations (CR14, 15) and five M. incognita populations (CR1, 3, 11, 12 and 17). The remaining range values of all characters overlapped among populations of the different species studied. Average values and observations of certain male characters are presented in Table 5. The ranges are presented in Table 17 in the appendix. All males had areolated lateral fields, although to a variable degree. In most populations they had four lines in the lateral fields, although five were also observed in some specimens of CR14, a population of M. hapla . Only one gonad was observed in males of the undescribed species (CR2) , the M. incognita populations CR3 and 11, and the M. hapla population CR15; the others had a varying percentage of males with two gonads. Highly significant differences were found among populations in the stylet, DEGO and spicules (chord of arch). The mean stylet length of the undescribed species was the lowest, followed only by those of M. exigua (CR7, 9), which had stylets 2.4 and 2.9 um longer, respectively. The stylet range of the undescribed species (CR2) overlapped with those of M. exigua (CR7, 9), while these two did not overlap with the range values of the M. incognita populations CR1, 3, 5, 6, 16 or 17. The ranges for the DEGO and spicule measurements overlapped among the different populations.

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27 Table 5. Morphological characters of males of 16 populations of Meloidogyne spp. from Costa Rica. Spicules Number of % males Pop. Stylet (chord of ar ch) DEG0* Areolation lateral lines with gpe gonad M. incognita *** PD 1 li.b I jj.u ni 3.5b yes 4 95 CR3 25.1 ni 34.5 ghi 3.0 ab yes 4 100 CR5 24.1 fg 32.6 efg 2.8 a yes 4 65 CD. U ghi 34.2 fghi 2.9 a yes 4 85 CR11 22.0 e 33.5 fghi 2.8 a yes 4 100 CR12 22.6 e 3.0 ab yes 4 80 OA / 24.4 fgh 32.3 ef 3.2 ab yes 4 70 CR17 25.7 J 1 35.4 i 3.5 b yes 4 95 M. exigua CR7 18.4 b 24.1 a 3.2 ab yes 4 45 CR9 18.9 D oc n a ZD . u a 4.9 de yes 4 85 M. hapla CR10 21.7 de 30.0 cd 5.0 e yes 4 60 CR14 20.4 c i3 . U DC 4.1 c yes 4-5 95 CR15 20.5 c 27.7 b 5.0 e yes 4 100 M. arenaria CR4 24.3 fgh 33.0 efgh 3.1 ab yes 4 95 M. sp. CR2 16.0 a 27.2 b 3.3 ab yes 4 100 M. incognita & M. hapla CR13 21.0 cd 28.2 be 4.4 cd yes 4 85 CV (%) 6.8 9.3 16.9 DEGO refers to the distance between the base of the stylet knobs and the dorsal esophageal gland orifice. ** *** The rest of the males had two gonads. k Mean of 20 observations. All measurements in um. Means in the same column followed by the same letter do not differ significantly from one another according to Duncan's Multiple Range Test (P = 0.01).

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28 Differential Plants Similar responses were obtained in the four replicates of each differential plant-population combination, and the average values are presented in Table 6. The differential plant responses indicated that populations CR1, 5, 6, and 17 were M. incognita race 1, populations CR3, 11, 12, and 16 were M. incognita race 2, population CR4 was M. arenaria race 2, populations CR7 and 9 were M. exigua , populations CR10, 14, and 15 were M. hapla , population CR2 was a different species, and population CR13 was a mixture of M. incognita and M. hapla . The responses to the differential plants gave evidence of pathogenic variation in M. exigua . The two populations of this species could be differentiated by their ability or inability to infect tomato, cv. Rutgers (Table 6). Population CR9 was able to reproduce well on this host but CR7 was not. A major difference among populations of M. hapla was found in the reaction of pepper, cv. California Wonder. Populations CR14 and 15 reproduced abundantly on this host, but CR10 reproduced only to a limited extent. Tomato was heavily infected and received the maximum rating value of 5 with all but the undescribed species (CR2) and the M. exigua population CR7. Tobacco was not infected by the undescribed species (CR2) and M. exigua (CR7, 9), only slightly by the M. incognita populations CR5, 6, and 17, and heavily by the remaining populations. Pepper was not a host for the undescribed species (CR2), and was infected only slightly by CR10, a population of M. hapla ; the other populations reproduced well on this host. Cotton was not infected, except slightly by CR6 and 16, two populations of M. incognita . Peanut was a good host for

PAGE 39

29 cm r-~ in • • • • o o CM VO • • O O — i cm m m CM m~crcnintninmin moo CM oooooooo o o m m in CM m in oooooooo o o o o o • inm^a-inmcoinm m m m *t h m m o o m vr m in m mmmmminmin m in m m * K C 00^ o o 0 cn _ pi h n ic m VO — I r— I i— ( i— I pi pi pi pi pi pi u u o o c_> o c o o c CO 43 a c SI co >h en <3cm °a —i pi a • a E B 01 •n u •W cn CO cu a; CO CO B CO CO •U CO CO O i o OJ u 60 oo u CO CD a) u CO a CO CO a co . — i T3 e CU c u M CO CO rH 0) 4J CU 4-4 CU 0 o u 0 c H B cu CO • * •H o o u *-> o c 60 1 o C 1 — 1 •H cn c T3 o >-< II •H O •U o -i CU I a, 4-J CU CO u 3 iH II j= CO 00 > cn •H cu • #> X. CO o l a) . — 1 CO 1 c cn •a o cu p. II u CO c CU CM CU Pi -a • rCM > • 1 CU CO . — l cu CO u II CO CO u . — i •H 1 — 1 4-J a. CO •H cu CU iH CO H

PAGE 40

30 M. hap la (CR10, 14, 15), moderate for the mixture of M. incognita and M. hapla (CR13) and a poor host for CR6, a population of M. incognita . Watermelon was not infected by the undescribed species (CR2) , two populations of M. hapla (CR14, 15), and only lightly by the third population of M. hapla (CR10) and by the mixture of M. incognita and M. hapla . This plant was a good host for the other populations. Finally, corn was not a host for the undescribed species (CR2) , M. exigua (CR7, 9), and for two populations of M. hapla (CR10, 14), a poor host for M. arenaria (CR4) , the mixture of M. incognita and M. hapla (CR13) , one population of M. hapla (CR15) and two of M. incognita (CR5, 17), and a good one for the other populations. Discussion Morphology M. incognita . The general shape of the perineal patterns was similar among the populations studied and could be used to distinguish this species from the others. The interpretation of the characteristics exhibited by the perineal patterns agreed with the reports by previous authors in Costa Rica (Hidalgo and Lopez, 1980a; Salazar and Lopez, 1980). Similarly, the absence of a posterior protuberance was noted. It was noticed that when the mean values of the morphometric characters of each population were compared to those previously reported from Costa Rica, the juvenile length and the female stylet in CR1 were greater. In CR3 the juveniles were wider; CR5 had juveniles with a greater length and females with a longer stylet; CR6 had longer juveniles, with longer tails and a greater a ratio; population CR11 had longer juveniles, and the interphasmidial distance, tail length, stylet base to head end, DEGO and the a ratio in

PAGE 41

31 the females were slightly greater. They also had a smaller anal width. In the females, the excretory pore was longer. Males of all populations had mean values of their characteristics similar to those reported by these authors (Hidalgo and Lopez, 1980a; Salazar and Lopez, 1980). M. exigua . There are no previous reports about the morphometries of this species in Costa Rica, so comparisons were made to the data provided by Chitwood (1949), Lordello and Zamith (1958), Esser et al. (1976), and Whitehead (1968). The general shape and the characteristics of the striae of the perineal pattern agreed with previous descriptions. A few females of each Costa Rican population had the neck region located on the ventral side of the body and a posterior protuberance. This observation contradicts the statement of Esser et al. (1976) and that of Whitehead (1968), who denied the presence of such protuberance on females of this species. On the other hand, some males of the two populations from Costa Rica had twisted bodies and some untwisted bodies. This observation was in agreement with the previous report by Scotto la Massese (1969), and contradicts the statement by Lordello and Zamith (1958), that males of M. exigua did not have a twisted body, thus constituting an exception among the root-knot nematodes. Another contradiction with the report by Lordello and Zamith (1958) was the finding of only one testis in some males of both populations. Lordello and Zamith (1958) reported that all males possessed two testes. The two Costa Rican populations had longer infective second-stage juveniles and females with longer stylets than previously reported.

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32 Males of CR7 had greater DEGO values than those reported by Lordello and Zamith (1958). All other values found in this investigation agreed with, and in some cases were identical to, those previously reported. M. hapla . The finding of this species outside the Central Plateau and the Central Volcanic Range, the only areas where it had been found previously (Lopez and Salazar, 1978; Mattey and Lopez, 1978; Lopez and Azofeifa, 1981; Salazar and Lopez, 1980) widens its reported geographical distribution in Costa Rica. Both El Empalme and Division are high altitude areas with high precipitation and relatively cool temperatures all year round. This agrees with the observed tendency for the distribution of M. hapla in the rest of Costa Rica (Lopez and Salazar, 1978). The shape of the perineal patterns and the characteristics of their striae were in close agreement with previous reports from Costa Rica (Lopez and Salazar, 1978; Salazar and Lopez, 1980), except that no wings were observed in the perineal patterns. Females of the three populations had greater values for the excretory pore and the DEGO than those reported for other Costa Rican populations (Lopez and Salazar, 1978; Salazar and Lopez, 1980). The population CR10 had greater values for the total length and tail length of the juveniles, and for the stylet and spicules (chord of arch) of the males. The CR14 population had longer spicules than found by previous authors. The other characters had mean and range values similar to those reported earlier (Lopez and Salazar, 1978; Salazar and Lopez, 1980). The recta of all juveniles were undilated. When first found in Costa Rica, Lopez and Salazar (1978) observed some juveniles with dilated recta in a population collected from cabbage. Later, these

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33 authors (Salazar and Lopez, 1980) found M. incognita and M. hap la coexisting in cabbage in the same general area of their first finding. Since M. incognita juveniles have dilated recta (Chitwood, 1949), the possibility of a mixture of both species in the first report seems likely, and therefore makes the report of dilated recta in M. hap la juveniles doubtful. M. arenaria. The finding of a population of M. arenaria in Ciudad Neilly is the first report of this species in Costa Rica. Comparisons were made to the values and observations given by previous authors (Chitwood, 1949; Esser et al., 1976; Eisenback et al., 1981). The general shape and characteristics of the striae of the perineal pattern were similar to those reported by these authors, but the second-stage juveniles were shorter than the value given by Eisenback et al. (1981), although similar to the values given by Chitwood (1949). All other morphometric values for females, males and juveniles were similar to the reports by the previously mentioned authors. Meloidogyne sp. n. The females of this root-knot nematode could be differentiated from the other species by the presence of a posterior protuberance and the neck and head regions located on the ventral side of the body. The body was usually oval, in contrast to the pyriform shape found in M. exigua , some of which showed a posterior protuberance and the neck on the ventral side of the body. Some, but not all specimens of M. exigua exhibited these same characters. The shape of the perineal pattern was also unique. Other differentiating characters were the short interphasmidial distance in the

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34 females, the longer juvenile tails, the smaller c ratio of juveniles and the shorter male stylet. The phasmids of the females were also smaller than in other root-knot nematode species. Differential Plants As pointed out by Sasser and Carter (1982), differential plants 1) provide a preliminary or corroborative indication of the root-knot nematode species being evaluated, based on the usual response of the hosts, and 2) detect pathogenic variation of a population, as determined by host responses different from the usual for the various species. However, differential plants cannot be relied upon entirely for identification, because the population being studied may be a mixture of species or a species for which no or limited host response data are available. For example, the undescribed species from rice did not reproduce on any of the differential plants (Table 6). The reaction of the plants, however, was used to differentiate among the other species studied, and even for the determination of the host race among populations of M. incognita and M. arenaria . Based on the scheme provided by Sasser and Carter (1982), the M. incognita populations CRl, 5, 6, and 17 were designated as race 1, whereas populations CR3, 11, 12, and 16 were designated as race 2. This is the first report of the presence of race 2 in Costa Rica. Salazar and Lopez (1980) had previously reported race 1 only. In spite of the evidence of pathogenic variation in the two populations of M. exigua , it seems premature at this time to call them races. This term was applied to populations of Meloidogyne species that were shown by numerous experiments to have unique host preferences, and that

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35 were named only after there was evidence of wide geographical distribution and/or sufficient significance for crop rotation and/or plant breeding programs (Taylor and Sasser, 1978; Sasser and Carter, 1982). Most of the criteria used for the application of the term host race were not fulfilled in this case. Future work could give the necessary proof that they indeed deserve to be designated as host races. From a practical point of view, this finding could be of value to farmers in the area of Sarchi, as some fields where coffee was grown were changed to tomato production. Differences in the ability of M. hap la populations to reproduce on pepper were found. Reactions of the other differential plants to the three populations were similar, and agreed with the usual response given by them to this species (Taylor and Sasser, 1978; Sasser and Carter, 1982). As in the case of M. exigua , it seems premature at this time to apply the term host races to these populations. The population of M. arenaria , similar to most of the populations in the world collection of the International Meloidogyne Project (Sasser and Carter, 1982), did not reproduce on peanut, cv. Florunner, and therefore was determined to be race 2 of this species. The reaction given by the differential plants to CR 13, the mixture of M. incognita and M. hap la , was different from the usual one given to each of the major species (Sasser and Carter, 1982). Previous workers have found this same mixture of species in the Central Volcanic Range of Costa Rica, on plants such as cabbage, carrot, lettuce and green peas (Lopez and Azofeifa, 1981; Padilla and Lopez, 1979; Perlaza et al., 1978; Perlaza et al., 1979; Salazar and Lopez, 1980). As pointed out by several of them, such mixture of species makes the management of

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36 root-knot nematodes by crop rotation and resistant cultivars even more difficult, and might require some long term studies for the development of profitable management schemes.

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CHAPTER III DESCRIPTION OF Meloidogyne salasi sp. n. Introduction In 1968, a root-knot nematode causing severe damage on upland rice ( Oryza sativa L.) was found in Volcan de Buenos Aires, Puntarenas, Costa Rica. The parasite was tentatively identified as a new species of Hypsoperine (Figueroa, 1973). Although several aspects of the biology, morphology and the pathogenicity of this nematode on rice were studied, no species description was given. In late 1979, high population densities of an undescribed root-knot nematode were found on rice, cv. C.R.1113, at La Cuesta, Puntarenas, Costa Rica (Alvarado and Lopez, 1981). This nematode caused severe damage on rice growing in the field and also was highly pathogenic to rice grown under greenhouse conditions. The nematode was localized on a few farms in the southeastern part of the country (Sancho, 1981). In addition, a root-knot nematode was reported infecting rice in the province of Cocle, Panama (Tarte, 1981) . Because of the severe damage caused by the nematode, farmers in the region abandoned rice production in favor of grasslands. Again, no description of the species was given (Tarte, 1981). An examination of a few perineal patterns from the population studied by Figueroa (1973) and some preserved specimens from Panama provided by Ing. Julio Lara, confirmed that the species involved was the same as the root— knot nematode found on rice at La Cuesta, Costa Rica. 37

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38 Populations of this root-knot nematode from both Costa Rica and Panama were studied cytologically (Triantaphyllou, 1982); both populations reproduce by obligatory mitotic parthenogenesis and both have a diploid chromosome number of 36. The nematode is described, illustrated and named herein as Meloidogyne salasi sp. n., in honor of Professor Luis Angel Salas Fonseca, the founder of Plant Nematology in Costa Rica. Materials and Methods A culture of M. salasi sp. n. , increased and maintained on rice, cv. C.R.1113, was established from eggs and infective second-stage juveniles obtained from the type locality of La Cuesta, Costa Rica. Nematodes from this culture were used for all morphological studies. Light Microscope (LM) Studies Galled rice roots were cut open in shallow petri dishes containing ® distilled water. Eggs were placed on a glass slide, ringed with Zut and covered with a cover slip. Other eggs were left overnight in the petri dish and the freshly hatched second-stage juveniles were placed ® ten per slide in a drop of water contained in a ring of Zut and a coverslip applied. Approximately 20 minutes after mounting the juveniles were observed and measured using a camera lucida. Males were dissected from old galls and prepared using the same method as described for the juveniles. Females were dissected from galled roots boiled in lactophenol for two minutes. The perineal patterns were prepared according to the method described by Franklin (1962) and modified by Taylor and Netscher (1974). Whole females were mounted on a cavity slide and their length (excluding neck) and maximum body width were drawn. The females were removed from

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39 the solution and punctured near mid-body with a fine needle to release the internal pressure. The head and the neck were excised and mounted in a drop of lactophenol on a slide, a cover slip added and ringed with ® Zut . Type specimens of males and females were fixed in 3% formalin for 48 hours, transferred to lactophenol at 50°C for 24 hours, and mounted in dehydrated glycerin. Nomarski differential interference optics was used to observe all specimens. Photomicrographs of males, second-stage juveniles and the perineal patterns of females were taken with an Olympus OM-2 camera. Drawings of males, females and second-stage juveniles were prepared with a camera lucida. Scanning Electron Microscopy (SEM) Studies Males, females and second-stage juveniles were processed for SEM by a modification of the techniques described by Eisenback and Hirschmann (1979, 1980), and Eisenback et al. (1980). Freshly hatched second-stage juveniles were obtained from eggs placed in distilled water for 18-24 hours at room temperature. The juveniles were transferred to 0.5 ml of distilled water, chilled at 5°C for one hour, and killed by adding three drops of cold (5°C) 4% glutaraldehyde solution buffered with 0.1 M sodium cacodylate at pH 7.1. More buffered 4% glutaraldehyde was added at 24-hour intervals, three drops at a time, until a final 2% concentration was obtained. Fixation continued for an additional 72 hours at 5°C. The juveniles were washed two times in sodium-cacodylate buffer (pH 7.1), transferred to a small plastic chamber with a fine screen (15 ym diameter pore) on the bottom and kept for 24 hours at 5°C. Postfixation was done with 2% osmium tetroxide, buffered with 0.1 M sodium cacodylate at pH 7.1, for 18-24

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40 hours at room temperature. This solution was replaced with sodiumcacodylate buffer and kept at 5°C for another 24 hours. Specimens were dehydrated with a graded series of ethanol (5-20-20-35-50-65-95-100%) at room temperature, with 24-hour intervals per step. Another screen was placed on top of the chamber, and the entire contents critical-point dried with CO 2 in a Balzer drier. Dried nematodes were position with one third of their body (the anterior or the posterior part) lying across a human hair that was placed on the surface of a stub covered with double-coated tape. They were coated with gold for five minutes in a Giko Engineering 1 B-2 model ion coater and viewed with a Hitachi S-450 scanning electron microscope operated at 20 KV of accelerating voltage. Type 55 Polaroid film was used for photomicrographs. Males were dissected from galled rice roots and treated as described previously for second-stage juveniles. Small pieces of galled roots containing females of M. salasi sp. n. were fixed in a 4% glutaraldehyde solution buffered with 0.1 M sodiumcacodylate at pH 7.1. After 6-7 days whole females were dissected from the roots and prepared as described previously for juveniles. In describing the external morphology of males, females, and secondstage juveniles, the terminology proposed by Eisenback and Hirschmann (1979, 1980) and Eisenback et al. (1980) was followed. Species Description Meloidogyne salasi sp. n. Females . Measurements of 50 females in lactophenol are presented in Table 7. Measurements of holotype in glycerin . Body length (excluding neck) : 422 um; maximum body width: 306 ym; neck length: 133 um; neck width at

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41 Table 7. Measurements of 50 females and eggs of Meloidogyne salasi sp. n. from rice, cv. C.R.1113. Character Mean Range Standard error of the mean Standard deviation CV (%) Female linear measurements (ym) Body length 486 .3 372. 0625 .0 8.92 63. 10 12.9 Maximum body width 338 . 1 209. 0425 .0 6.61 46. 76 13.8 Neck length 135 . 1 86. 0203 .0 3.25 22. 99 17.0 Neck width at middle of metacorpus 63 .3 43. 799 .9 1.33 9. 41 14.8 Middle of metacorpus to head end 78 .2 60. 999 .9 1.43 10. 16 12.9 Metacorpus width 35 .7 29. 041 .8 0.39 2. 79 7.8 Metacorpus length 35 . 6 30. 043 . 4 0.44 3 . 12 8.7 Metacorpus valve width 10 .6 9. 013 .7 0.13 0. 94 8.9 Metacorpus valve length 13 .7 11. 515 .6 0.13 0. 97 7.0 Stylet 10 .0 8. 112 .5 0.11 0. 84 8.4 Stylet knobs height 2 . 1 1 . 53 . 4 0.05 0. 41 19.3 Stylet knobs width 3 .4 2. 54 .5 0.06 0. 44 12.9 DEGO 4 .9 3. 46 .8 0.14 . 1. 00 20.3 Excretory porehead end 5l i . i 1 8 7 . j i / i i n n c Zo *3 1 Q Vulva slit length 21 .9 15. 926 .5 0.34 2. 43 11.0 Anus-vulva 16 .4 9. 024 .0 0.41 2. 94 17.9 Interphasmidial distance 15 .2 10. 621 .8 0.33 2. 35 15.4 Female ratios a 1 .4 1. 02 .0 0.02 0. 20 14.2 Body length/neck length 3 .7 2. 15 .8 0.12 0. 85 23.1 Stylet knobs width/height 1 .6 0. 82 .6 0.05 0. 36 22.1 Metacorpus length/width 1 .0 0. 71 .2 0.01 0. 09 9.8 Metacorpus valve length/width 1 .3 0. 91 .5 0.01 0. 11 8.7 Egg linear measurements (pm) Length 94 .5 82. 8113 .2 0.74 5. 20 5.5 Width 41 .1 38. 244 .5 0.22 1. 50 3.8 Egg ratios Length/width 2 .3 1. 92 .7 0.01 0. 13 5.8

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42 middle of metacorpus: 43.8 pm; middle of metacorpus to head end: 71.9 ym; metacorpus width: 30.5 pm; metacorpus length: 33.6 pm; metacorpus valve width: 9.5 pm; metacorpus valve length: 12.2 pm; stylet: 10.9 pm; stylet knobs height: 2.1 pm; stylet knobs width: 3.2 pm; DEGO: 4.5 pm; a ratio: 1.37; body length/neck length: 3.17; stylet knobs width/height: 1.52; metacorpus length/width: 1.10; metacorpus valve length/width: 1. 28. Female as in general description. Perineal region not visible. Description (Figs. 4, 5, 6, 7, 8, 9, 10, 11). Body pearly white, with body length (excluding neck) /maximum body width (ratio a) with an average value of 1.4 and a range of 1 to 2. Distinct posterior protuberance present (Figs. 4, 5). Neck inserts on the ventral side of body, its position varying from approximately even with anterior end of body to about one-third of body length ventrad to this point. Center line of neck and axis of body (straight line from middle of perineal area to the anterior most part of body) making an angle that varies between 20 and 130°. Cuticle distinctly annulated, often with incomplete annulations in the head and neck regions. Head region offset from body. In SEM (Fig. 6A-D) the labial disc appears slightly elevated, with the rounded and relatively large prestoma located in the middle. The labial disc and the medial lips form an anchor-shaped structure, with the ventral lip (determined from the position of the excretory pore) being pointed. In a few cases the ventral lip is not pointed, but the anchor-shaped structure is still recognizable (Fig. 6D) . Inner labial sensillae are difficult to see. Head region appears as a single annule, often marked by longitudinal lines. Amphid openings are clearly distinct, rectangular. Lateral lips are arched, slightly larger than the labial disc. Cephalic framework has lateral sectors larger than ventral or dorsal sectors.

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43 of females of Meloidogyne salasi

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44 Fig. 5. Female of Meloidogyne salasi sp. n. .

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Fig. 6. Scanning electron photomicrographs of face views of females of Meloidogyne salasi sp. n. .

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46

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47 The vestibule and vestibule extension are clearly distinct when observed with the LM (Fig. 7). Stylet is delicate, and the cone is usually straight, with a triangular base about 1/4 of its length, tapering to a fine, pointed tip. Opening of stylet is near the tip, in the anterior 1/4 of the cone. The shaft has approximately the same diameter throughout and is shorter than the cone. Stylet knobs offset from the shaft, and are ovoid to almost triangular in shape. Lumen of stylet in the stylet knobs is about the same as in the procorpus, but it narrows sharply in the stylet cone. Outlet of the dorsal esophageal gland is branched twice, with dorsal ampulla relatively large. Excretory pore position is relatively variable, about 1-1% times the stylet length behind the stylet knobs in 66% of the specimens observed. In a few females (4%) the excretory pore was about 1/2 stylet length behind the stylet knobs, whereas in others (6%) it was about 3 times the stylet length behind the stylet knobs. Lumen of esophagus is strongly sclerotized in the procorpus and metacorpus, but difficult to see beyond the latter. Metacorpus is relatively large and rounded (Fig. 8), with a strong, oval central valve. Esophageal glands appear as a massive, globose structure with five nucleated lobes, which are often difficult to distinguish. Nuclei are difficult to observe with bright field illumination but distinct with Nomarski differential interference optics. Perineal pattern is oval-shaped (Figs. 9, 10, 11) and has fine outer striae and somewhat coarse striae in the inner portion. The striae are mostly unbroken, smooth, relatively few in number and far apart. Perineum has no or only one striae, and only a few in the roughly circular central area of the pattern. Vulva is a transverse, smooth slit, with no or few striae coming out of its sides. Phasmids are small,

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48 Fig. 7. Cephalic region of a female of Meloidogyne salasi sp. n. .

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49 Fig. 8. Anterior region of a female Meloidogyne salasi sp. n. .

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Fig. 9. Photomicrographs of female perineal patterns of Meloidogyne salasi sp. n. . A, B and C from Costa Rica. D from Panama.

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51

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52 Fig. 10. Perineal patterns of Meloidogyne salasi sp. n.. A, B and C from Costa Rica. D from Panama.

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53 Fig. 11. Scanning electron photomicrographs of female perineal pattern of Meloidogyne salasi sp. n. .

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54 closely spaced. Dorsal arch is high and wide, usually rectangular in shape, but somewhat square in some specimens. There is no evidence of lateral lines or interrupted striae. Tail tip is prominent in freshly mounted perineals. Punctations are lacking. Males . Measurements of 50 males in distilled water are presented in Table 8. Measurements of allotype in glycerin . Body length: 1,711 ym; maximum body width: 35.3 ym; body width at base of knobs: 16.6 urn; body width at excretory pore; 26.6 ym; body width at middle of metacorpus: 22.2 ym; excretory pore to head end: 136.7 ym; middle of metacorpus to head end: 94.5 ym; head height: 5.3 ym; head width: 10.9 ym; excretory pore to middle of metacorpus: 48.4 ym; esophageal lobe end to head end: 243.2 ym; stylet: 19 ym; stylet base to head end: 21.8 ym; stylet shaft plus knobs: 9 ym; stylet cone: 10 ym; stylet knobs height: 2.3 ym; stylet knobs width: 3.3 ym; DEGO: 4.4 ym; metacorpus width: 11.9 ym; metacorpus valve width: 3.4 ym; metacorpus valve length: 8.4 ym; testis: 1,034 ym; testis %: 60.4; spicules: 28.8 ym; gubernaculum: 9 ym; tail length: 13.8 ym; cloaca-phasmids : 8.4 ym; ratio a: 48.4; ratio b: 7.0; ratio c: 123.9. Description (Figs. 12, 13, 14). Vermiform, with variable body length, tapering at the anterior end (Figs. 12A, 12B) and relatively rounded at the posterior end (Figs. 12C, 12D) . Head region slightly offset from body, bearing a variable number of incomplete annulations, with distinct head cap (Figs. 12A.B, 13A.B). In SEM the large, rounded labial disc is slightly elevated above the medial lips, with lateral edges slightly arcuate (Figs. 14A,B). The oval prestoma is in the center of the labial disc, encircled by six inner labial sensillae which have

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55 Table 8. Measurements of 50 males of Meloidogyne salasi sp. n. from rice, cv. C.R.1113. Standard Standard error of deviCharacter Mean Range the mean ation CV (%) Linear measurements (ym) Total length 1 ,619 .0 992 .02,093 40.87 289. ,04 17.8 Maximum body width 33 .9 25 .441 Q . o 0.49 3. ,53 10.3 Body width at base of knobs 16 .8 11 .8on 7 . / 0.19 1. 40 8.3 Body width at exc. pore 26 .8 23 .1J4 0.33 2. 40 8.9 Body width at middle of metacorpus 23 .6 20 .127 .0 0.22 1. 58 6.6 Exc. pore to head end 156 .9 88 .0227 .0 4.56 32. ,31 20.5 Middle of metacorpus to head end 101 .7 64 .0134 .0 2.61 18. ,51 18.2 Head height 4 .5 2 .55 .6 0.09 0. 69 15.3 Head width 10 .4 7 .513 . 1 0.15 1. 12 10.7 Exc. pore to middle of metacorpus 55 .8 18 .799 .9 2.89 20. 44 36.6 Stylet 18 .2 12 .121 .8 0.31 2. 19 12.0 Stylet base to head end 20 .6 15 . 923 . 1 0.27 1 . 96 9.5 Stylet shaft + knobs 10 .4 6 .812 .5 0.20 1. 43 13.7 Stylet cone 7 .7 4 .310 .3 0.17 1. 22 15.7 Stylet knobs height 3 .1 2 .14 .2 0.07 0. 54 17.6 Stylet knobs width 4 . 6 3 .57 .5 0. 09 0. 66 14.1 DEGO 4 .1 2 .85 .9 0.10 0. 72 17.4 Metacorpus width 12 .6 8 .416 .2 0.24 1. 73 13.7 Metacorpus valve width 5 .1 3 .17 .1 0.12 0. 88 17.2 Metacorpus valve length 6 .8 4 .88 .7 0.14 0. 99 14.4 Testis 887 .1 353 .01,250 25.24 178. 51 20.1 Spicules 25 .8 17 .534 .5 0.63 4. 52 17.4 Gubernaculum 7 .8 5 .611 .8 0.19 1. 34 17.0 Tail length 13 .0 6 .539 .0 0.65 4. 66 35.7 Cloaca-phasmids 4 . 1 0 .39 .9 0.35 2. 50 59.6 Phasmids-tail end 8 .8 4 .017 .5 0.36 2. 58 29.1 Ratios a 47 .5 31 .858 .1 0.92 6. 51 13.6 c 132 .8 46 .6-: 254 .7 5.46 38. 62 29.0 Body length/middle of metacorpus to head end 16 .0 11 .721 .6 0.32 2. 33 14.4 Head region width/height 2 .3 1 .83 .0 0.03 0. 26 11.4 Stylet knobs width/height 1, .5 1, .03, .0 0.04 0. 32 21.2 Metacorpus valve length/width 1 .3 0 .72 .3 0.04 0. 30 21.7 Percentages Excretory pore 9. .7 6, ,512. ,7 0.20 1. 43 14.7 Testis 55, .0 32, ,071, ,6 1.11 7. 90 14.3

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Fig. 12. Males of Meloidogyne salasi sp. n.. A Esophageal region (ventral). B Cephalic region (lateral). C,D Tail (lateral).

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Fig. 13. Anterior region of males of Meloidogyne salasi sp. n. . A,B Scanning electron microscope photomicrographs. C,D Light microscope photomicrographs.

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58

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Fig. 14. Scanning electron photomicrographs of males of Meloidogyne salasi sp. n.. A,B Face views. C Lateral field. D Tip of spicules showing pores.

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i

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61 pitlike openings. Stoma has a slitlike opening. Medial lips are wider than the labial disc, forming a continuous head cap with it, with no discernible indentations at the lateral junctions. Four cephalic sensillae appear as slight, small cuticular depressions on the medial lips, two on each. Amphidial openings are relatively long slits below the lateral edges of the labial disc. Lateral lips are almost inconspicuous, and marked by short grooves that start near the lateral junction of the medial lips and the labial disc, and extend into the head region. One to three rows of short, incomplete annulations are present at different levels of the head region (Figs. 13A,B). Frequently the specimens have one row on one side and two or three on the opposite side. Cuticle has distinct annules, about 1.9 um wide near the head region, 2 um wide around the middle of the body and 1.6 um wide near the tail. Lateral field is about 6, 7.5 and 5 um wide near the anterior, middle portion and tail areas of the body, respectively. There are usually four lines in the lateral field, one at each edge of the ridge and two in the inner portion, but in some specimens five or up to six lines are visible for some distance in the middle of the body; the additional lines are fainter. Lateral fields start as two lines with crenate edges near the base of the stylet, some four to 10 annules behind the head region, continue posteriorly as far as the middle of the procorpus, where the inner two lines appear, and continue to the posterio end. The tail portion is twisted about 90°. The lateral fields are areolated in their entirety, and usually correspond with the body annulations, but in some areas, especially the middle portion, they do not (Fig. 14C). The cephalic framework is sclerotized, and its lateral sectors are slightly larger than the head cap (Figs. 12A; 13C.D).

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62 Stylet is robust, with a pointed cone, slightly longer than the shaft. The cone has the opening near the tip and a triangular base in the basal h of its length. Stylet shaft is of same diameter throughout, with ringlike structure near its base (Figs. 12A, B) . Stylet knobs are rounded, offset from the shaft, with an ascending slope toward base of stylet shaft (Figs. 12B; 13C,D). Lumen of stylet is almost as wide as that of the procorpus, but narrows at the cone. Outlet of the dorsal esophageal gland has two branches, with a relatively small dorsal ampulla. Procorpus is two to three times as long as the muscular, elongated, oval metacorpus (Fig. 12A) . The metacorpus has a strongly sclerotized central valve. Nerve ring encircles the short isthmus. Distinct excretory pore, with long, curved excretory duct that disappears as it approaches the intestine. Basal lobe of esophagus overlaps the intestine ventrally and has three nuclei, with the anterior nucleus near beginning of lobe and posterior nucleus near the end of the lobe. Hemizonid 1-2 annules long, located 1-2 annules anterior to excretory pore. Intestinal caecum extends anteriorly on dorsal side of body to about the level of the nerve ring. Most specimens have one outstretched testis, but it may be reflexed for a short distance. A few specimens have two testes. If two testes are present, one may be outstretched and the other reflexed, but both of about the same length. Sperm are globular, granular. Spicules are long, arcuate, typical of the genus (Figs. 12C.D). In SEM each spicular tip shows one transverse opening (Fig. 14D). Gubernaculum is simple. Phasmids are located typically below the cloacal opening, with a pore-like opening. Body twists about 90° near the cloacal region. Second-stage juveniles . Measurements of 50 juveniles in distilled water are presented in Table 9.

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63 Table 9. Measurements of 50 second-stage juveniles of Meloidogyne salasl sp. n. from rice, cv. C.R.1113. Standard Standard error of deviCharacter Mean Range the mean ation CV (%) Linear measurements (um) Total length 464. 4 422503 2. 59 18. 35 3. 9 Esophageal lobe base to head end 121. 8 102 i-153 1. 31 9. 26 7. 6 Maximum body width 16. 2 15. 319 .3 0. 11 0. 83 5. 1 Body width at excretory pore 15. 0 13. 115 .9 0. 59 0. 64 4. 2 Middle of metacorpus to excretory pore 23. 6 16. 831 .5 0. 46 3. 27 13. 8 Middle of metacorpus to head end 56. 7 50. 662 . 1 0. 36 2. 58 4. 5 Head region width 6. 2 5. 07 .8 0. 08 0. 58 9. 2 nsaa region nexgnu T J • •3 J 1. 85 .6 n 1 9 fi7 o / Q o Stylet 11. 4 9. 213 .3 0. 15 1. 07 9. 3 Stylet cone 5. 2 3. 76 .8 0. 11 0. 83 15. 8 Stylet knobs width 2. 3 1. 52 .8 0. 04 0. 30 13. 2 Stylet knobs height 1. 5 1. 02 . 1 0. 03 0. 22 14. 8 Stylet base to head end 14. 7 12. 116 .2 0. 09 0. 69 4. 6 Stylet shaft 4. 7 2. 86 .2 0. 09 0. 70 14. 8 DEGO 3. 7 2. l5 .3 0. 08 0. 58 15. 5 Metacorpus valve length 3. 9 2. 85 . 3 0. 06 0. 45 11. 4 Metacorpus valve width 3. 4 2. 54 .3 0. 04 0. 33 9. 7 Excretory pore to head end 80. 3 71. 589 .6 0. 57 4. 03 5. 0 Tail length 67. Q 0 56. 580 .2 (J . 1 i r J . lb / . b Tail terminus length 19. 7 11. 826 .2 0. 47 3. 33 16. 8 Tail terminus width at beginning 5. 1 3. 76 .2 n 09 n -J Anal width 11. 8 10. 715 .0 0. 10 0. 71 6. 0 Anus-beginning of terminus 47. 9 38. 158 .7 0. 67 4. 77 9. 9 Ratios a 28. 6 23. 932 .2 0. 24 1. 73 6. 0 b 3. 8 3. 04 .4 0. 04 0. 30 7. 9 c 6. 8 5. 97 .7 0. 05 0. 42 6. 1 Tail length/anal width 5. 7 4. 26 .8 0. 07 0. 54 9. 4 Tail length/tail terminus length 3. 5 2. 45 .7 0. 08 0. 58 16. 7 Head region width/height 1. 9 1. 22 .8 0. 05 0. 37 19. 3 Stylet knobs width/height 1. 5 0. 72 .1 0. 03 0. 25 16. 7 Metacorpus valve length/width 1 . 1 0. 61 .5 0. 02 0. 16 13. 9 Percentages Excretory pore 17. 2 16. 018 .7 0. 08 0. 61 3. 5

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64 Fig. 15. Second-stage juveniles of Meloidogyne salasi sp. n. A Esophageal region (lateral) . B Cephalic region (lateral). C Tail (dorsal). D Tail (lateral).

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65 Fig. 16. Scanning electron photomicrographs of face views of secondstage juveniles of Meloidogyne salasi sp. n. .

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66 Fig. 17. Lateral field of second-stage juvenile of Meloidogyne salasi sp. n. .

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Fig. 18. Second-stage juveniles of Meloidogyne salasi sp. n A,B Anterior region. C,D Tail terminus.

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68

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69 Description (Figs. 15, 16, 17, 18). Body is vermiform, tapering at both ends but much more so posteriorly (Figs. 15A,B,C,D). Head region slightly narrower than the body, and elevated head cap (Figs. 15B) . In SEM the elongated labial disc is slightly elevated above the medial lips, with lateral edges straight or almost so (Fig. 16A). Oval prestoma in the center of the labial disc, encircled by six inner labial sensillae with pit-like openings. Stoma with a small slit-like opening. Medial lips crescentic in most specimens, wider than the labial disc, with no discernible indentations at the lateral junctions with it, forming a dumbell-shaped cap. In a few specimens one of the medial lips is pointed (Fig. 16B). Amphidial openings are slitlike, and located below the lateral edges of the labial disc. Lateral lips are narrow, with straight or slightly arcuate edges, almost parallel to the lateral edges of the labial disc. Head region is smooth, without annulations. Cephalic framework is weakly developed. Body is distinctly annulated, the annulations being discernible with the LM up to the beginning of the tail terminus. Lateral fields are areolated, with four lines, the external two are slightly crenate (Fig. 17.). Anteriorly the exterior two lines begin at about the middle of the procorpus, followed by one and finally two interior lines (Fig. 17). All four lines continue past the anus, where the central lines disappear and the two exterior ones continue for a short distance to the beginning of the tail terminus. The stylet is weakly developed, and has small, rounded knobs, one slightly larger and in a lower position than the other two (Figs. 15A,B; 18A.B). The knobs have an ascending slope toward the shaft. A ring-like structure encircles the shaft near its base (Fig. 15B) . Ampulla of the dorsal gland duct near its opening into the lumen of the esophagus.

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70 Procorpus is about 2-2% times as long as the muscular, oval metacorpus which has a sclerotized central valve. Nerve ring encircles the narrow isthmus. Hemizonid located 1-2 annules anterior to the excretory pore, about 1 annule long. Excretory pore located at about the same level or slightly posterior to the nerve ring. The curved excretory duct disappears as it approaches the intestine. Basal lobe of esophagus rather short, with three nuclei, the anterior one located near its beginning and the posterior one near its end. The basal esophageal lobe overlaps the intestine ventrally (Fig. 15A) . Anal opening is a small pore on the cuticle. Rectum is weakly dilated. Tail relatively long, tapering to a fine, rounded, slightly clavate terminus (Figs. 15C,D). Eggs . Measurements of 50 eggs in distilled water are presented in Table 7. Description . Eggs similar to those of other species of the genus, and are enclosed in a soft, highly water-soluble gelatinous matrix. Up to 2,000 eggs/egg mass have been counted on galled rice roots collected from the type locality (L. Salazar, personal communication). Diagnosis : M. salasi sp. n. is closely related to the recently described M. kralli (Jepson, 1983), and also to M. acronea (Coetzee, 1956) and to M. graminis (Sledge and Golden, 1964). M. salasi sp. n. can be distinguished from M. kralli by the dimensions of the female (body length of 486 um vs 463 urn, maximum body width of 338 um vs 306 um) , the straight shorter stylet (10 um vs 13.1 um) , longer excretory pore of the female (32 um vs 15.8 um) , by the higher dorsal arch of the perineal pattern, and absence of a postero-laterally directed irregular double incisure on either side of the tail region of the perineal pattern, longer males (1,619 um vs 1,076 um) , greater a and

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71 c ratios in the males (47.5 and 132.8 vs 31.7 and 117, respectively), shorter stylet cone in the male (7.7 ym vs 9.5 ym) , longer excretory pore in the males (156.9 ym vs 127 ym) , areolation of the lateral fields, annulations in the head region of the male (up to 3 vs 1), position of the phasmids on the male (posterior to cloaca vs at level of cloaca) . Additional differentiating characters in the second-stage juveniles include the body length (464 ym vs 439 ym) , the smaller a and b ratios (28.6 and 3.8 vs 31 and 6.5, respectively) and the smaller tail/anal width ratio (5.7 vs 7). M. salasi sp. n. can be distinguished from M. acronea by the female body length (486 ym vs 980-1,040 ym) , maximum body width (338 ym vs 530750 ym) , shorter stylet in the female (10 ym vs 12 ym) , shorter spicules of the male (25.8 ym vs 33-35 ym) , longer phasmids-tail end distance (8.8 ym vs 4 ym) , areolation of the lateral fields in the male, shorter second-stage juveniles (464 ym vs 490 ym) , smaller a, b, and c ratios in the juveniles (28.6, 3.8, and 6.9 vs 32, 5.4, and 9.4, respectively), longer tail of the juveniles (67.8 ym vs 49 ym) and longer tail terminus (19.7 ym vs 3.5 ym) . Finally, M. salasi sp. n. can be distinguished from M. graminis by the body length of the female, maximum body width and DEGO (486, 338, and 4.9 ym vs 726, 472, and 3.7 ym) , respectively), the absence of lateral lines in the perineal pattern, the fine striae in the perineal pattern; in the males by the greater a ratio (47.5 vs 43.5), the smaller c ratio (132 vs 187), the longer DEGO (4.1 ym vs 2.4 ym) , the longer tail (13 ym vs 8.4 ym) and the areolation of the lateral fields in the second-stage juveniles by the length (464 ym vs 475 ym) , the smalle a ratio (28.6 vs 31.7), the greater b ratio (3.8 vs 2.3), the longer DEGO (3.7 ym vs 2.4 ym) , the shorter esophageal lobe to head end distance

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72 (121.8 pm vs 200 pm) , the shorter tail (67.8 pm vs 78 pm) and the greater tail/anal width ratio (5.7 vs 4.3). Host range ; M. salasi sp. n. did not infect any of the plant species used in the North Carolina Differential Host test (Taylor and Sasser, 1978). Greenhouse studies conducted in Panama (Tarte, 1981) showed that Cynodon plectostachyus , £. dactylon , Ischaemutn cilia re , Digitaria decumbens , Tripsacum laxum , Echinocloa polystachya , Leucaena leucocephala , Kazungula sp . , Brachiaria ruziziensis , J3 . zuazilandensis , B_. rugulosa , Panicum maximum and Saccharum sinensis are poor hosts for this nematode. Figueroa (1973) reported that Homolepis aturensis is a host. The grass Echinocloa colonum is a host under field conditions at the type locality (R. Lopez, unpublished data). Holotype (female) : Isolated from greenhouse culture derived from original population obtained at La Cuesta, Costa Rica. Slide M-39, Nematode collection, Laboratorio de Nematologia, Facultad de Agronomia, Universidad de Costa Rica, San Jose, Costa Rica. Allotype (male): Same data as holotype. Slide M-13, Laboratorio de Nematologia, Facultad de Agronomia, Universidad de Costa Rica, San Jose, Costa Rica. Paratypes (males, females and second-stage juveniles): Same data as holotype. USDA Nematode collection, Beltsville, Maryland. Type host and locality : Rice ( Oryza sativa L.), cv. C.R. 1113, from La Cuesta, province of Puntarenas, Costa Rica.

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CHAPTER IV VARIABILITY OF Meloidogyne incognita FROM FLORIDA Introduction Prior to 1949 several investigators reported variations in populations of root-knot nematodes [then known as Heterodera marioni (Cornu, 1979) Goodey, 1932] parasitizing different plants. The first such record is apparently that of Sherbakoff (1939). He described root-knot disease on upland cotton growing on land where the previous crop was cotton. But he noted that in another field upland cotton was not infected when the previous crop was tomato infected with root-knot nematodes. Variation in host specificity on peach to root-knot nematode populations were reported by several authors (Day and Tufts, 1940; Christie and Havis, 1943). Evidence of the existence of physiological variation within R. marioni was also given in other reports (Christie, 1946; Christie and Alvin, 1944; Reynolds, 1949). In 1949, the genus Meloidogyne was reestablished and separated from Heterodera Schmidt, 1971 (Chitwood, 1949). Five species and one variety were described, viz. M. exigua , M. javanica , M. incognita , M. hapla , M. arenaria and M. incognita var. acrita. Sasser (1954) devised a scheme for the identification of the five species and one variety described by Chitwood (1949) based on the ability of each to infect certain plant species. This method was successful in identifying species that occurred in a given region, but it was not reliable when the species came from different geographical regions 73

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74 (Sasser, 1966; Sasser, 1972). In these instances the differential hosts may not give what has been called the "usual response for each species" (Sasser, 1972). Results of several hundred differential host tests on populations from all over the world were used as the basis to separate four host races within M. incognita (Sasser, 1979; Sasser and Carter, 1982). These race designations are based on the ability to infect and reproduce on cotton, cv. Deltapine 16, and tobacco, cv. NC-95. Race 1 does not parasitize or reproduce on either, whereas race 2 reproduces readily on tobacco. Race 3 reproduces on cotton but not on tobacco, and race 4 reproduces on both. According to Sas ser and Carter (1982), the consistency of host response to the four races, the widespread occurrence, especially of races 1, 2 and 3, and the number of populations involved shows that these races are stable taxa. The four races are as yet indistinguishable morphologically and are apparently unrelated to the cytological races distinguished in this species (Triantaphyllou, 1981). Schemes other than that of Sasser (1979) have been used to distinguish races of M. incognita . The degree of reproduction on soybean and alfalfa varieties was used by Boquet et al. (1975) and by Goplen et al. (1959). Other authors used the amount of root necrosis, root galling and the capability to parasitize sweetpotato varieties to distinguish races of M. incognita and M. incognita var. acrita (Giamalva et al., 1963; Martin and Birchfield, 1973). Fox and Miller (1973) distinguished two races of M. incognita by the number of egg masses produced on five differential hosts. They concluded that the number of galls was not a reliable index of the reproduction of root-knot nematodes.

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75 Populations of M. incognita in Tennessee differ considerably in pathogenicity (Southard and Priest, 1973). Six races were distinguished among 17 isolates evaluated on six differential hosts. In Florida, Perry and Zeikus (1972) found variation in the response of four populations of M. incognita to differential hosts. One population collected from strawberry reproduced well on this host, whereas another population collected from tomato was the only one that reproduced on pepper, cv. California Wonder. A third population, collected from sugarcane, reproduced on tobacco, cv. NC-95; the others did not reproduce on this host. Finally, a population collected from peach was the only one to reproduce on 'Okinawa' peach rootstock. Kirby (1972) reported variation in host preference among 14 populations of M. incognita collected from nine counties in Florida. Some populations caused severe galling on cotton, cv. Coker 201, whereas most did not. Variations in pathogenicity on sweetpotato, cv. Puerto Rico, also were noted. Only one population caused galling on tobacco, cv. NC-95. More recently, Lopez and Dickson (1977) found no differences in host reactions among three populations of M. incognita from Florida; all were identified as race 1. When Chitwood (1949) reestablished the genus Meloidogyne , he used the characteristics of the female perineal pattern, the stylet of secondstage juveniles, males and females, and the location of the dorsal esophageal gland orifice to distinguish the five species and one variety he described. Dropkin (1953) concluded that the general shape and perhaps other characters of the perineal pattern are under hereditary control.

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76 Variability in populations originating from a single juvenile of M. incognita var. acrita was less than in populations with a mixed ancestry. Allen (1952) demonstrated the variability in perineal patterns of M. incognita var. acrita originating from single egg mass isolates and grown on three plant species. Triantaphyllou and Sasser (1960) observed that in many isolates of M. incognita var. acrita the perineal patterns varied from typical acrita to typical incognita type. They stated that the morphological distinction between the subspecies was often uncertain, and that since the perineal pattern gives little information on the behavior of a given population, the division of the species into two subspecies had no practical purpose. They suggested that all populations with perineal patterns ranging from incognita to acrita-type be considered one species, M. incognita . Esser et al. (1976) however, considered that some differences in the coarseness of the striae in the perineal pattern, the dilation of the rectum, the a and c ratios of second-stage juveniles and the spicules of the male were solid enough features to give M. acrita the status of species. Riggs and Winstead (1959) were able to correlate the ability of certain isolates of M. incognita var. acrita and M. incognita to parasitize resistant tomatoes with certain morphological characters, i.e., patterns with more arch and longer second-stage juveniles. These differences, however, were not enough to exclude any of the new strains from the parental type. Priest and Southards (1971) could not morphologically distinguish six races of M. incognita among 16 populations of this species. Significant differences were noted in total length,

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77 c ratio and stylet length of second-stage juveniles among certain populations. Kirby (1972) found variation among 14 populations of M. incognita from Florida in mean vulva length, female interphasmidial and anus-vulva distances, DEGO of males and total length, stylet base to head end, maximum body width, tail length, DEGO and a and c ratios of second-stage juveniles. Range values of all measurements overlapped among populations. Lopez and Dickson (1977) reported statistically significant differences among three populations of M. incognita race 1 in several morphometric parameters. These parameters were stylet and DEGO of females, stylet, DEGO and spicules (chord of arch) of males, and total length, stylet base to head end, tail length, maximum body width, anal width and a and c ratios of second-stage juveniles. In spite of these differences, no individual character could be used to distinguish among the three populations since the ranges of all measurements overlapped. This study was conducted to characterize three populations of M. incognita from Florida by morphology and the reaction of differential plants. Materials and Methods Nematode Populations The designations and original sources of three Florida populations of M. incognita were: M-165, tobacco, Alachua County, 1974; M-195, corn, Suwannee County, 1977, and M-198, tomato, Manatee County, 1980. From several dozen egg masses these populations were increased on tomato, cv. Rutgers, in a greenhouse. A mixture of soil and builders sand (3:1 v/v, pH 7.1, 1.5% O.M., 91.8:6.2:2.0% sand: silt: clay) treated with steam at

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78 100°C for 24 hours was used in all tests. Each pot was fertilized twice a week with 100 ml of a 1% solution of Nutrisol® (12-10-20). Differential Plants Seven differential plants were inoculated with each population, and the plant responses were evaluated 60 days later. The differential plants used were identical to those used in Costa Rica: corn, cv. Minnesota A-401, tobacco, cv. NC-95, pepper, cv. California Wonder, cotton, cv. Deltapine 16, peanut, cv. Florunner, watermelon, cv. Charleston Grey, and tomato, cv. Rutgers. Pregerminated seeds of all plants, except tobacco, were prepared. Plants were inoculated at the following ages: corn-10 days old; peanut, cotton and watermelon-15 days old; tomato-30 days old, and tobacco and pepper-50 days old. The inoculum consisted of eggs and a few secondstage juveniles that were collected using a 1.05% sodium hypochlorite solution (Hussey and Barker, 1973). Approximately 10,000 eggs and or second-stage juveniles were pipetted over 1,000 cm 3 of soil in clay pots. Individual seedlings were planted and 500 cm 3 of soil added. Four replications of each differential plant-population combination were placed randomly on a greenhouse bench and kept separated by plastic dividers. The pots were fertilized twice a week with 100 ml of 1% Nutrisol® fertilizer solution (12-10-20) during the first five weeks of plant growth. Sixty days after inoculation the root systems were removed and immersed in a 0.0016% Phloxine B solution for one hour (Dickson and Struble, 1965) to stain the egg masses. Each root system was rated using the following scale (Taylor and Sasser, 1978):

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79 0: 0 egg masses 3: 11-30 egg masses 1 : 1-2 egg masses 4: 31-100 egg masses 2: 3-10 egg masses 5: more than 100 egg masses. Perineal patterns were prepared whenever it was judged appropriate to verify the identity of the females reproducing on a particular differential plant. For each character, 20 specimens in each population were observed. Al measurements were taken from outlines drawn with a camera lucida at 1.000X magnifications, except second-stage juvenile length (100X) and female length and maximum body width (40X) . Females were dissected from galled tomato roots boiled in lactophenol for two minutes. The perineal patterns were prepared according to the technique developed by Franklin (1962) and modified by Taylor and Netscher (1974). The perineals were divided into zones, following the method of Esser et al. (1976). Whole females were mounted in lactophenol on a cavity slide and their length and maximum body width drawn. The females were removed from the solution and punctured near mid-body with a fine needle to release the internal pressure. The neck and head regions were excised and mounted in a drop of lactophenol on a glass slide, covered with a ® coverslip and ringed with Zut . To obtain second-stage juveniles, egg masses were collected randomly from galled tomato roots and kept for 18-24 hours in distilled water. Live juveniles were mounted in a drop of water on a glass slide ® ringed with Zut and covered with a coverslip. Fifteen to 20 minutes later the juveniles were observed under a light compound microscope, Morphology

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80 certain characters were outlined and the position of the hemizonid in relation to the excretory pore and the dilation of the rectum were noted and recorded. To obtain males, galled tomato roots were dissected in petri dishes containing distilled water. After one to two hours the males were mounted using the same technique as described for the juveniles. Results Differential Plants Populations M-195, M-165 and M-198 were identified as M. incognita race 1, race 2 and race 3, respectively (Table 10). Tomato, cv. Rutgers, pepper, cv. California Wonder, watermelon, cv. Charleston Grey, and corn, cv. Minnesota A-401, were good hosts for all M. incognita populations. Peanut, cv. Florunner, was not a host for any population. Morphology The interpretation of the predominant type of perineal pattern for each population is presented in Table 11. None of the populations had a posterior protuberance, lateral incisures or striae in the perineum. A few striae on the vulva of population M-165 were observed coming out of its sides, but no such striae were present in M-195 or M-198. The three populations had relatively few striae in zone 1. The striae of populations M-195 and M-198 in zones 2, 3 and 4 were wavy, broken and relatively few in number, whereas those of M-165 were wavy and broken, but relatively moderate in number. The general shape of the perineal patterns in the three populations was pyriform, with a high trapezoidal dorsal arch. Some slight variants from this predominant shape were observed. An illustration of one perineal pattern from each population is presented in Fig. 19.

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81 Table 10. Response of seven differential plants to three populations of Meloidogyne incognita from Florida. Population Plant M-195 M-165 M-198 Tomato 'Rutgers' Tobacco 'NC-95' Pepper 'California Wonder' Cotton 'Deltapine 16' Peanut 'Florunner' Watermelon 'Charleston Grey' Corn 'Minnesota A-401' * 5 0 5 0 0 5 4.5 race 1 5 5 5 0 0 5 4.5 race 2 5 1 5 5 0 5 4.5 race 3 Average of four replicates. Response evaluated according to the number of egg masses/root system. 0=1; 1 = 1-2; 2 = 3-10;3 = 11-30; 4 31-100 and 5 = >100 egg masses.

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82 Table 11. Interpretation of the predominant type of perineal pattern of females of three host races of Meloidogyne incognita from Florida. Character M-195 race 1 Population M-165 race 2 M-198 race 3 Posterior protuberance * A A a A Vulva lip striae A F A Perineum A A A Lateral incisures A A A Striae Zone 1 F F F Zone 2 FWB MWB FWB Zone 3 FWB MWB FWB Zone 4 FWB MWB FWB Shape pyrif orm , with a high trapezoidal dorsal arch. A = absent; F = Few; M = moderate in numbers; W = wavy; B = broken.

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83 Fig. 19. Photomicrographs of female perineal patterns of three populations of Meloidogyne incognita from Florida.

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84 The average values of the female morphometric characters are presented in Table 12. No significant differences in excretory pore, stylet, anus-vulva and interphastnidial distances were found among populations. Population M-195 had a significantly shorter DEGO than M-165 and M-198, whereas the latter had a significantly shorter body length than M-195 and M-165. The maximum body width of M-165 was significantly greater than those of M-195 and M-198. The vulva of M-165 was significantly longer than that of M-198. The range of these measurements are presented in Table 18 of the appendix. The mean values of the characters of second-stage juveniles are presented in Table 13, and the ranges in Table 19 of the appendix. No significant differences among populations were found in maximum body width, anal width and stylet base to head end. Population M-198 had significantly shorter total length and tail length than populations M-165 and M-195. This last population had significantly longer DEGO than the other two. The difference in DEGO between M-165 and M-198 was significant also. The a ratio was significantly different for each population. The c ratio of M-198 was significantly greater than those of M-165 and M-195. The second-stage juveniles of the three populations had dilated recta, and the hemizonid was located anteriorly to the excretory pore. Morphological observations and the average values of some morphometric characters of the males are presented in Table 14. Range values for these characters are presented in Table 20 in the appendix. All males observed had four lateral lines, areolated lateral fields, and in all populations 95% of them had one gonad. No significant differences

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85 Table 12. Comparative morphological data (um) from females of three host races of Meloidogyne incognita from Florida. Character M-195 race 1 Population M-165 M-198 race 2 race 3 CV (%) Excretory pore 26.0 a 23.8 a 27.4 a 35.0 Stylet ** DEGO 16.5 a 16.0 a 16.8 a 7.1 4.3 b 4.9 a 5.1 a 17.4 Maximum body width 441.0 a 535.0 a 463.0 b 12.0 Body length 716.0 a 715.0 a 641.0 b 12.8 Vulva slit length 21.9 ab 22.7 a 20.9 b 8.5 Anus-vulva 16.1 a 16.3 a 15.6 a 9.2 Interphasmidial distance 18.3 a 19.7 a 18.2 a 15.3 Mean of 20 observations. Means in horizontal rows followed by the same letter do not differ significantly according to Duncan's Multiple Range Test (P = 0.01). * DEGO refers to the distance between the base of the stylet knobs and the dorsal esophageal gland orifice.

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86 Table 13. Comparative morphological data (pm) from second-stage iuveniles of three host races of Meloidogyne incognita from Florida. Character Population M-195 M-165 M-198 CV (%) race 1 race 2 race 3 Total length 430.0 a 431.0 a 399.0 b 3.5 Tail length 54.0 a 55.4 a 43.0 b 5.8 Maximum body width 15.0 a . 15.4 a 15.1 a 4.0 Anal width 10.5 a 10.8 a 10.7 a 5.6 Stylet base to head end 15.3 a 15.2 a 15.1 a 2.9 ** DEGO 2.4 b 2.9 a 3.1 c 9.6 a 28.8 b 27.9 a 26.2 c 4.5 c 8.0 a 7.7 a 9.2 b 5.8 Rectum dilation yes yes yes *** Hemizonid anterior anterior anterior * Mean of 20 observations. Means in horizontal rows followed by the same letter do not differ significantly according to Duncan's Multiple Range Test (P = 0.01). ** DEGO refers to the distance between the base of the stylet knobs and the dorsal esophageal gland orifice. *** Position in relation to the excretory pore.

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87 Table 14. Comparative morphological data (urn) from males of three host races of Meloidogyne incognita from Florida. Population Character M-195 M-165 M-198 CV (%) race 1 race 2 race 3 Stylet * 23.1 a 23.6 a 23.4 a 5.6 Spicules (chord of arch) 28.5 a 31.0 a 29.9 a 14.0 ** DEGO 2.9 b 3.2 ab 3.4 a 16.6 Areolation yes yes yes Number of lateral lines 4 4 4 *** % males with one gonad 95 95 95 * Mean of 20 observations. Means in horizontal rows followed by the same letter do not differ significantly according to Duncan's Multiple Range Test (P = 0.01). ** DEGO refers to the distance between the base of the stylet knobs and the dorsal esophageal gland orifice. *** The rest of the males had two gonads.

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88 among populations were found in stylet and spicules (chord of arch) . Population M-195 had a significantly shorter DEGO than M-198. Ranges of all measurements and ratios for females, second-stage juveniles and males overlapped among the three populations. Discussion The finding of race 1, 2, and 3 of M. incognita confirmed previous reports (Araujo et al. , 1983; Kirby, 1972; Lopez and Dickson, 1977) that at least three host races of M. incognita exist within the state of Florida. However, there was a small difference in the reaction of one of the differential plants. Corn was rated as a poor host for the M. incognita populations studied by Kirby (1972) and Lopez and Dickson (1977), but a good host for the populations used in this study. Difference in the corn cultivar and the M. incognita populations used in these studies, as well as in the rating scales used to evaluate the response of this differential plant could explain this difference. When differential plant responses were compared to those of the Costa Rican populations, no differences were noted, except that race 3 of M. incognita was not detected from Costa Rica. Several statistically significant differences among the three populations studied were found in certain morphometric characters. It could be postulated that there is a relationship between some of these characters and the ability of a population to parasitize a given differential host. For instance, one could associate host race 3 with a shorter female body length and vulva, and a shorter second-stage juvenile with shorter tails, smaller a ratio and greater c ratio and DEGO values. Race 2 could be associated with a larger female body width, and intermediate DEGO and a ratio values in the second-stage juvenile. Race 1

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89 could be associated with a shorter DEGO in the females, second-stage juveniles and males, and a greater c ratio in the second-stage juveniles. This association, however, has a weak basis as the ranges of all measurements and ratios overlapped among populations, making it difficult to distinguish them. A somewhat similar situation was found by Hirschmann (1981). She analyzed the external morphology of second-stage juveniles and males of 14 populations of M. incognita belonging to two (A and B) cytological races with the SEM. All four host races were represented among the populations studied. No correlation between morphology and host race was found. Some populations in each cytological race appeared to be distinct, but still shared the general features characteristic of the species. When the morphometric data gathered in this study were compared to those of previous studies in Florida, some similarities and differences were noted. The general shape of the perineal patterns, the characteristics of their striae, the absence of posterior protuberance, lateral incisures and striae in the perineum and vulva length of the females were similar to the reports by Kirby (1972) and Lopez and Dickson (1977). Similarities were found also in stylet base to head end, DEGO, maximum body width, anal width, rectum dilation and anterior location of the hemizonid in relation to the excretory pore in second-stage juveniles, and in areolation, number of lateral lines, stylet and DEGO of males. The a and c ratios, and the tail length of second-stage juveniles had similar values, in most cases, to those reported earlier, except that M-198 had a smaller a ratio, a shorter tail and a greater c ratio. Differences observed included slightly shorter interphasmidial and anus-vulva distances in the females, longer female DEGO, slightly longer

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90 female stylet and maximum body width of second-stage juveniles. In M-195 and M-198 the spicules (chord of arch) were shorter, and in M-195 and M-165 the second-stage juveniles were longer, whereas they were shorter in M-198, as compared to the previous reports of populations of this species from Florida (Kirby, 1972; Lopez and Dickson, 1977). As in previous comparisons, some differences and some similarities were found when the data from the Floridian populations were compared to M. incognita populations from Costa Rica. The male spicules (chord of arch), as well as the female interphasmidial distance, had smaller values than the populations from Costa Rica. The female stylets were slightly longer in the Floridian populations, whereas the vulva and the anusvulva distance were slightly shorter. Similarities included the absence of a posterior protuberance in the female body, as well as the lack of vulva lip striae, perineum striae and lateral incisures in the perineal patterns, the presence of a few striae in zone L, the character of the striae in zones 2, 3, and 4 of the perineals, the excretory pore and the maximum body width and body length of the females. The DEGO of the females were also similar, except that in M-198 it was slightly longer. In males, the DEGO, stylet, areolation of the lateral fields and number of lateral lines were also concordant to those from Costa Rica. The maximum body width, anal width, stylet base to head end, anterior location of the hemizonid with respect to the excretory pore and rectal dilation of the second-stage juveniles were also similar to those of the Costa Rican populations. The DEGO was similar in M-165 and M-198 but slightly shorter in M-195. The tail was shorter in M-198 and similar in the other two populations. The a ratio was greater in M-198 than in the populations from Costa Rica. Finally,

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91 the total length and the c ratio of the juveniles were in some cases concordant, in others smaller and in others greater than the ones found in the Costa Rican populations.

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CHAPTER V ELECTROPHORETIC PATTERNS OF SOME Meloidogyne spp. FROM COSTA RICA AND FLORIDA Introduction The variability of many morphological characters, and the presence within species of so-called physiological races, are two of the most important problems associated with the taxonomy of plant parasitic nematodes (Allen and Sher, 1967). These problems have prompted the seach for other approaches not based entirely on anatomy and morphology, to aid in the indentif ication and characterization of species and races of nematodes (Hussey, 1982). Among these approaches, biochemical systematics is one which has provided new and helpful information about nematodes and their phylogenetic relationships, complementing and extending the information provided by classical morphologically-based taxonomy (Hussey, 1979). Biochemical systematics exploits the subtle molecular differences that underlie taxonomic variation (Hansen and Buecher, 1970). As suggested by Hussey (1982), the ultimate goal of taxonomy should be the classification of the genotypes of the organisms. Practical methods for the analysis of the nucleotide sequence in genes are not currently available. Proteins, on the other hand, are an expression of the sequence of the nucleotides in a gene, and the analysis of these molecules may provide an approach for comparing the genotypes of nematodes. 92

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93 Electrophoretic techniques allow the separation and identification of specific soluble enzymes and other nonenzymatic proteins. Many enzymatic proteins have different molecular forms, with identical or similar substates, called isozymes (Siciliano and Shaw, 1976). Electrophoretic comparisons of the isozyme patterns obtained from individual specimens or mass homogenates of a population, can give an idea of the similarity between different populations (Nei and Chakraborty, 1973). The objectives of this study were to use starch gel electrophoresis to a) differentiate several species of root-knot nematodes found in Costa Rica, b) compare four populations of M. incognita race 2 from Costa Rica to three host races of this same species from Florida, and c) investigate possible differences among three host races of M. incognita from Florida. Materials and Methods Nematode Populations Ten populations of Meloidogyne spp. from Costa Rica and three of M. incognita from Florida, U.S.A., were studied. These populations had already been identified based on their morphology and reactions on plant differentials (Chapters II and IV). The Costa Rican populations used were M. salasi sp. n. (CR2) , M. arenaria race 2 (CRA) , M. exigua (CR7, 9), M. hapla (CR10, 14), and M. incognita race 2 (CR3, 11, 12, 16). The three populations of M. incognita from Florida were M-195 (race 1) , M-165 (race 2) and M-198 (race 3). Most populations were increased on tomato, cv. Rutgers. M. exigua was increased on pepper, cv. California Wonder, and M. salasi sp. n. was increased on the wild grass Echinocloa colonum . A mixture of soil and builders sand (3:1 v/v, pH 7.1, 1.5% O.M.,

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94 91.8:6.2:2.0% of sand: silt: clay) treated with steam at 100°C for 24 hours was used to grow the plants. Each pot was fertilized twice a week during the first five weeks of growth with 100 ml of a 1% solution of ® Nutrisol (12-10-20). The pots were placed randomly on a greenhouse bench and kept separated from other populations by plastic dividers to avoid contamination. Sample Preparation Sixty days after inoculation, the root systems were cut into sections approximately 3 cm long and treated as described by Hussey (1971), except for a few modifications. These modifications consisted of agitating the flasks containing chopped roots in .200 ml of Pectinol 59L at 150 oscillations per minute for 18-24 hours, the use of a 1.0 M sucrose solution for the centrifugation of the suspension of females, and the transferring of the females directly from the 60-mesh sieve to a beaker with a 1% NaCL solution. The females were collected free of debris, with a Pasteur pipette. Twenty females of each nematode population, except M. exigua , were placed in polyethylene microcentrifuge tubes (7x30 mm, 250 yl) , the saline solution was removed and replaced with 10 pi of a 0.1 M K 2 HP0 4 buffer with 0.8% of NaCl and 0.001 M MgCl 2 (Hussey et al., 1972). M. exigua samples were comprised of 40 females. Samples were stored at 85°C with no detectable loss of enzymatic activity noted in most samples stored up to eight months. Starch Gel Electrophoresis Horizontal starch gel electrophoresis was performed on equipment modified from Bush and Huettel (1972). The modification consisted of using a plexiglass U-shaped mold (15 cm long, 17.4 cm wide, 1 cm deep, with legs 1.5 cm long, 4 cm high and 17.4 cm wide for holding the starch

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95 gel. The bottom openings along each leg of the mold were taped shut and the mold was filled with starch gel solution. The tape along each leg was removed just prior to placing them into the electrode buffer (Steiner and Joslyn, 1979). The starch solution was prepared by adding a mixture of 47.5 g of hydrolysed starch (Connaught Laboratories Limited, Ontario, Canada) and 21 g of electrostarch (Otto Hiller Co., Wisconsin, U.S.A.) to 450 ml of the appropriate buffer for the protein system under analysis. Three buffer systems were used for the analysis of the different isozymes. C-buffer system (Ayala et al., 1972) was used for malate dehydrogenase (MDH) , isocitrate dehydrogenase (IDH), and malic enzyme (ME). CA-7 buffer system was used for a-glycerophosphate deydrogenase (a-GPDH) and glucose-6-phosphate deydrogenase (G-6-PDH) , whereas CA-8 buffer system was used for hexokinase (HK) (Steiner and Joslyn, 1979). Poulik and tris-ethylenediamine-tetracetic (EDTA) -borate buffer system was used for phosphoglucose isomerase (PGI) and fumerase (IBM) (Bush and Huettel, 1972). The nematode protein was prepared as described by Huettel et al. (1983), except that the grinding buffer consisted of 0.01 M trisma base, 0.001 M EDTA free acid in 500 ml of deionized water, pH 7.0. At 2.5 cm from the cathode end of the starch gel, two adjacent strips, 1 cm wide each, were cut with a metal spatula. The strip closer to the end was temporarily removed and the second strip was slid back into the space that was occupied by the first. The homogenate saturated wicks were blotted lightly and loaded on the vertical edge of the large portion of the gel, ca. 0.5 cm apart, starting from the lateral edge. The second strip of gel was then moved back to its original position, and the strip which had been temporarily removed was put into place.

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96 The gel was gently pressed back together, and placed inside a refrigerator at 5°C. Each gel was covered with plastic food wrap, a glass plate was put on top and then a metal container with ice, to which NaCl had been added, was placed on top of the glass plate (Bush and Huettel, 1972). The gels were electrophorized for 15 hours at 30 milliamps for the C-buffer system, 15 hours at 50 milliamps for the CA-7 buffer system, 6 hours at 30 milliamps for the CA-8 buffer system, and 6 hours at 50 milliamps for the Poulik and Tris-EDTA-borate buffer system. After electrophoresis gels were removed from the trays and sliced horizontally, that is, parallel to the surface, into four slices, 2 mm thick. The sites of isozyme activity were determined by immersing the slabs in the appropriate enzyme reaction mixtures. The mixtures described by Steiner and Joslyn (1979) were used for the detection of MDH, IDH, ME, a-GPDH , G-6-PDH, and HK. The reaction mixtures detailed by Bush and Huettel (1972) were used for the determination of PGI and FUM activity. The gel slabs were incubated in the dark at 37°C for 15-75 minutes depending upon the enzyme. Once stained, the slabs were removed from the reaction mixture, washed with running water and photographed. They were immersed in a fixing solution consisting of methanol, deionized water and glacial acetic acid (5:5:1, v/v) for 18 hours, washed again with running water, wrapped in plastic food wrap and stored in a refrigerator at 5°C. Two preliminary tests were conducted for each system with a few samples, and after obtaining consistent results, four gels were run with samples from each population for each of the systems studied.

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97 Results All isozymes resolved migrated anodically on the gels. The bands of each enzyme were numbered consecutively beginning with the one nearest the origin. Each enzyme is discussed separately, and the results mentioned constitute an average of the four observations performed in each case. The intensity of the staining reaction on the gel surface was related to enzyme activity. The results obtained with all of the enzymes are illustrated in Fig. 20. Malate Dehydrogenase M. salasi sp. n. showed four bands, with MDH1 appearing weakly stained at 25 mm from the origin. MDH2 produced a heavily stained band starting 38 mm from the origin. MDH3 and MDH4 appeared as two weakly stained bands which started 46 and 61 mm from the origin, respectively. In population CR7 of M. exigua two weakly stained bands were located 19 and 25 mm from the origin, whereas CR9, the other M. exigua population, had one weakly stained band which started 19 mm from the origin. Population CR10 of M. hapla had a heavily stained band which started 24 mm from the origin; there was evidence of a weakly stained, narrow band located 34 mm from the origin. CR14, the other population of M, hapla , had three bands; MDH1 was heavily stained and started 54 mm from the origin. MDH2 and MDH3 were weakly stained, and started 64 and 75 mm from the origin, respectively. M. arenaria (CR4) and M. incognita (CR3, 11, 12, 16, M-195, 165 and 198) had all three bands located at the same position. In all cases MDH1 was heavily stained and started 24 mm from the origin. MDH2 was weakly stained and started 34 mm from the origin. MDH3 was weakly stained and started 42 mm from the origin. With the

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98 PHOSPHOGLUCOSE ISOMERASE + FUMERASE + a GLYCEROPHOSPHATE DEHYDROGENASE ISOCITRATE DEHYDROGENASE MALATE DEHYDROGENASE 1 1 MM can n a 0 ORIGIN |2 |S |2 |S |S |S |S |S |S IS |S ? |S T3 b x |x |» l» I E 1 1 3 3 3 3 3 3 3 O O O o O 0 n o o o 0 o O o i ID D ID ID ID in 3 3 3 3 3 3 0) 0) 0) 0) 0) 0) 0) X 3J 3) > > > o o o m ™ in 33 33 X D > > > > o o o o m rn m m M N m m m !" . u . 20. Diagramatic sketch of comparative electrophoretic patterns of some Meloidogyne spp. from Costa Rica and Florida. Left to right: M. salasi sp. n. ; M. exigua (CR7) ; M. exigua (CR9) ; M. hap la (CR10) ; M. hap la (CR14) ; M. arenaria ; M. incognita (CR3) ; M. incognita (CR11); M. incognita (CR12); M. incognita (CR16); M. incognita (M-195) ; M. incognita (M-165) and M. incognita (M-198).

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99 exception of CR16, in which case the band stained heavily, all MDH bands were weakly stained. Isocitrate Dehydrogenase Population CR10 of M. hapla had two weakly stained bands which started 33 and 37 mm from the origin. CR14, the other M. hapla population, had three bands which started 44, 50 and 56 mm from the origin. The IDH3 band was heavily stained whereas IDH1 and IDH2 were weakly stained. M. arenaria (CR4) and M. incognita (CR3, 11, 12, 16, M-195, 165 and 198) all had three bands located at the same positions. IDH1, IDK2 and IDH3 started 25, 34 and 40 mm from the origin, respectively. With the exception of the three bands of M. arenaria , and IDH3 of the M. incognita population CR12, which were heavily stained, the other bands stained relatively weakly. Activity of this enzyme was not detected in homogenates of M. salasi sp. n. and M. exigua . a-glycerophosphate Dehyrogenases In M. salasi sp. n. there was one heavily stained band which started 24 mm from the origin. Population CR7 of M. exigua had one weakly stained band which started 13 mm from the origin. Population CR9 of M. exigua showed only one weakly stained, narrow band located 18 mm from the origin. Population CR10 of M. hapla had two weakly stained bands, which started 14 and 18 mm from the origin. CR14, the other M. hapla population, had one heavily stained band which started 17 mm from the origin. All populations of M. incognita had one band which started 11.5 mm from the origin. In CR3, 16, M-165 and 198 the band was heavily stained, whereas it was weakly stained in the other populations. Activity of this enzyme was not detected in homogenates of M arenaria.

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100 Fumerase One heavily stained band, starting 4 mm from the origin, was detected in M. salasi sp. n. . In M. exigua no activity of the enzyme was detected on two occasions, but when the number of females per samples was increased to 100, population CR7 showed one weakly stained band which started 9 mm from the origin. CR9 also had the same as described for CR7, except that it was very weakly stained. Population CR10 of M. hap la had one weakly stained band which started 8 mm from the origin. The other M. hapla population (CR14) had one heavily stained band which started 9 mm from the origin. All populations of M. incognita had one band, heavily stained, which started 4 mm from the origin. The activity of this enzyme could not be detected on homogenates of M. arenaria . Phosphoglucose Isomerase M. salasi sp. n. had one heavily stained band which started 18 mm from the origin. M. exigua had, in 3 out of 4 gels, one very weakly stained band which started 16 mm from the origin. Population CR10 of M. hapla had two heavily stained bands which started 11 mm and 16 mm from the origin, whereas population CR14 of this same species had only one heavily stained band, which started 10 mm from the origin. M. arenaria and all populations of M. incognita had one heavily stained band which started 16 mm from the origin. No activity was detected for G-6-PDH and HK, and no discrete bands were observed for ME, although some smearing was evident on all gels indicating enzyme activity.

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101 Discussion The starch gel electrophoretic analyses demonstrated certain enzymes could be used to differentiate among most of the species of Meloidogyne used in this study. Thus isozyme patterns provide and additional criterion for the taxonomic classification of these nematodes and confirms other electrophoretic studies with root-knot nematodes (Dalmasso and Berge, 1979; Dickson et al., 1971; Hishibashi, 1970; Hussey et al. 1972) . The MDH, IDH, a-GPDH, FUM and PGI patterns revealed intraspecif ic differences in M. exigua and M. hapla . Each population of these two species were collected from different localities in Costa Rica, and also different hosts in the case of M. hapla . Population CR7 of M. exigua reproduced on tomato to a limited extent, whereas CR9 reproduced well on this host. Some morphological differences between males of both populations were observed with the scanning electron microscope (R. Lopez, unpublished data). Regarding the two populations of M. hapla , certain morphological differences in several characters of the second-stage juveniles were noted also (Table 4). It seems likely that the populations of both M. exigua and M. hapla differed in their genetic composition. Therefore it is not surprising to have found differences in their enzyme patterns. Moreover, these electrophoretic differences could be considered as additional evidence that these populations are physiologically different. Other enzyme analyses should provide more confidence in their identification by enzyme profiles. The comparison of four populations of M. incognita race 2 from Costa Rica to three host races of this species from Florida, showed that no differences existed in any of the enzymes among these populations. Some slight differences were found in the intensity of the staining

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102 reactions of certain isozymes patterns, but they did not seem to be important enough to be considered diagnostic for a specific host race of M. incognita . Several factors may be responsible for the failure to detect enzymatic activity of G-6-PDH and HK, and for obtaining only smears in the ME gels. The method of culturing, stage of development, physiological state of the nematodes, protein extraction procedures, storage conditions of the protein extract and the method of protein analyses are factors that can induce variability in electrophoretic analyses of nematode proteins. These factors may influence the number of proteins or isozymes that can be detected, their electrophoretic mobility or both (Hussey et al., 1972). Some or all of these factors, or the interaction of some of them could be responsible for the unsatisfactory results. A similar explanation could be given regarding the failures to detect activity of IDH in homogenates of M. salasi sp. n. and M. exigua , and of a-GPDH and FUM in homogenates of M. arenaria . In any case, it seems desirable to invesigate these aspects, not only with the same populations used in this study, but with others of the same species, to determine whether the unsatisfactory results are due to the specific population or to the methodology. Those enzymes which did not appear consistently, or which appeared only after increasing the number of females in the samples, i.e., PGI and FUM in homogenates of M. exigua , may have concentrations that were too low to be suitable for starch gel electrophoresis. It is also possible that freezing affected the activity of these enzymes. While tomato was used as the host for most populations, pepper was the host for M. exigua and E^. colonum was the host for M. salasi sp. n. .

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103 Evidence indicated that enzyme profiles may be influenced by the plant on which the root-knot nematodes are increased (Hussey et al., 1972; Ishibashi, 1970; Starr, 1979). Dickson et al. (1971), however reported identical enzyme patterns for M. javanica isolated from different hosts. When possible it is desirable to propagate all of the nematode populations on a common host to avoid the possibility of their influence on enzyme profiles. The use of life stages other than the females, such as the egg or the freshly hatched second-stage juvenile, which are free from plant protein, might prove to be a more reliable subject for electrophoretic analyses as was found for some round cyst nematodes (Greet and Firth, 1977). Starch gel electrophoresis is a promising approach for the study of several specific enzymes in root-knot nematodes, and could be used, in addition to those based on morphology, responses of differential plants, cytology and mode of reproduction, for the taxonomic classification of this important group of plant pathogens.

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CHAPTER VI CONCLUSIONS Useful information for the nematologists, and in general for those dealing with agriculture in Costa Rica was obtained with the characterization of several local populations of the economically important rootknot nematodes. New data were obtained on the range and mean values of certain morphometric characters for most of the species currently identified in Costa Rica. Data were gathered also concerning their ability to reproduce on certain plant differentials. This information can be used in the identification of field populations and for the implementation of management strategies based on crop rotation and use of resistant cultivars, and aimed at reducing the damage induced by them. The results provide the basis for the first report of the presence of M. arenaria race 2 in Costa Rica. Evidence of pathogenic variation in populations of M. exigua and M. hapla also were obtained. For instance, one population of M. exigua reproduced well on tomato, whereas the other did not. A similar situation was found with three populations of M. hapla . Two populations reproduced abundantly on pepper, whereas the third reproduced only to a limited extent. These results may provide a basis for the future recognition of host races within both species. The known geographical distribution of M. hapla in Costa Rica was widened to localities outside the Central Plateau and the Central Volcanic Range. 104

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105 A root-knot nematode pathogenic to rice ( Oryza sativa L.), M. salasi sp. n., was described and illustrated. This nematode was found in the southeastern part of the country, and is also present in rice-producing regions of Panama. It can be distinguished from the related M. kralli , M. acronea and M. graminis by quantitative and qualitative characters of the males, females and second-stage juveniles. Morphometries and plant differential studies confirmed the presence of races 1, 2 and 3 of M. incognita in Florida, U.S.A.. When they were compared to several populations of this same species from Costa Rica, certain morphometric similarities as well as differences between them were found. These differences however, were not clear enough to be considered reliable characters for the differentiation of these populations. Starch gel electrophoresis showed differences in certain isozyme patterns among the different species studied. Malate dehydrogenase and phosphoglucose isomerase are considered particularly useful for distinguishing between M. incognita , M. hap la , M. arenaria , M. exigua and M. salasi sp. n. , and even within populations of both M. exigua and M. hapla . In general, there was good agreement between the morphology and starch gel electrophoresis analyses of isozymes regarding the identification and separation of the previously mentioned nematode species. In order to fully take advantage of the information provided by the biochemical approach for the identification of species and host races within this group, it is important to promote greater research emphasis on it.

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106 The results obtained by people working in this area should be combined with those of other investigators working in morphology, cytology and biology of the root-knot nematodes. The additional information would provide a more complete vision of this important group of plant pathogens.

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APPENDIX

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CO u cn o u I o M 4-1 pu CO cu c >> oo o "3 0) 2 to C O H •u cO H 3 a o Cu SO e o cn V H CO e a) c aj E CD M 3 cn co j CO 3 H C 3 < > CO 4-1 > 60 H C 3 0) > >H en CN r--. CN SO 00 cn 00 o m CO as m CO CO 00 00 CO o> CO 00 1 1 I 1 1 I 1 1 1 1 1 1 1 1 1 1 T3 c oo o CN CO .— i CO r-cn cn 00 00 V40 00 ~D O m CN 0> o> cn CQ I— 1 , x -a co o 2 .o o o w Q t-H 4-1 CO o 4J 0) 1-1 u w o PL4 o cn m CN i— i CM CN CN cn cn cn CN CN cn o CN cn o> 00 cn cn cn cn cn cn CM cn cn cn CM m I 1 1 1 r^. 1 r^1 r-1 1 1 1 m 1 I I 1 I 1 cn CN cn < cn cn cn o — l . — i • — i i — i •— H OS ptS os os OS os PS OS OS oS OS OS OS OS os U u o u u u u u u u u a u o u 108

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109 Table 16. Range of measurements of males from 16 populations of Meloldogyne spp. from Costa Rica. Population Stylet Spicules (chord of arch) DEGO CR1 21-26 28-38 2.1-5.2 CR2 14-18 24-32 2.6-4.2 CR3 23-27 27-38 2.1-4.2 CR4 21-26 26-40 3.1-3.1 CR5 21-26 24-39 2.1-3.6 CR6 23-26 26-39 2.1-4.2 CR7 16-20 21-26 2.1-4.2 CR9 16-20 21-28 4.2-6.3 CR10 20-23 26-32 4.2-6.3 CR11 20-25 25-39 2.1-3.6 CR12 20-27 26-37 2.1-4.2 CR13 20-27 25-41 3.6-7.3 CR14 18-23 25-32 3.6-5.2 CR15 20-22 25-29 4.2-5.2 CR16 22-27 26-37 2.1-5.2 CR17 24-26 28-42 3.6-4.2 All measurements in ym.

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110 o U w a CO C CO oooooooooocooot\Ovo ^Hf-H^— < i-H »-H i-H i— ( i-H i-H f— I t— ti — I t— I t— ( r-H i-H I I I I I I I I I I I I I I I I t^.r«»r^r»-oor-»f^sor^r>.r«.rs.vor>»r^r«. ^3-rO«*- u u v_> O U u u o

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Ill Table 18. Range of measurements of females from three host races of Meloidogyne incognita from Florida. Population Character M-195 M-165 M-198 race 1 race 2 race 3 Excretory pore 13 .152.5 13 .448.4 17. 244.1 Stylet 14 .419.1 12 .818.1 15. 019.7 DEGO 3 .15.0 3 .87.2 3. l6.9 Maximum body width 328 .0601.0 476 .0593.0 375. 0609.0 Body length 562 .0867.0 539 .01015.0 468. 0796.0 Vulva slit length 17 .825.9 20 .325.9 17. 224.7 Anus -vulva 11 .918.4 14 .417.8 13. 120.3 Interphasmidial distance 12 .226.3 16 .625.0 14. L24.1 * All measurements in pm.

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112 Table 19. Range of measurements of second-stage juveniles from three host races of Meloidogyne incognita from Florida. Population PI— LyD PI— 10 J pi— iy o race 1 race 2 race 3 Total length 406.0-469.0* 409.0-459.0 375.0-425.0 Tail length 44.157.5 50.060.3 36.946.6 Maximum body width 13.815.9 14.116.6 13.815.9 Anal width 9.711.6 10.311.9 9.712.2 Stylet base to head end 14.715.9 14.116.3 14.715.6 DEGO 2.23.1 2.53.4 2.73.8 a 27.0-30.7 25.230.6 24.029.2 c 7.5-10.0 7.38.4 8.410.7 * All measurements in pm.

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113 Table 20. Range of measurements of males from three host races of Meloidogyne incognita from Florida. Population Character M-195 M-165 M-198 race 1 race 2 race 3 Stylet 20.3-25.3* 20.9-26.3 21.6-25.0 Spicules (chord of arch) 17.2-35.0 25.3-37.2 23.4-36.9 DEGO 2.23.4 2.24.4 2.84.7 * All measurements in um.

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LITERATURE CITED Allen, M.W. , and S.A. Sher. 1967. Taxonomic problems concerning the phytoparasitic nematodes. Annual Review of Phytopathology 5:247-264. Alvarado, M. , and R. Lopez. 1981. Extraction de nematodos asociados al arroz, cv. C.R. 1113, mediante modif icaciones de las tecnicas de centrifugacion-f lotacion y embudo de Baermann modif icado. Agron. Costarr. 5:7-13. Alvarado, M. , and R. Lopez. 1982. Recuperacion de larvas de Meloidogyne incognita de tres suelos tropicales por modif icaciones de las technicas del embudo de Baermann modificado y centrifugacion-f lotacion. Turrialba 32:83-87. Araujo, M.T., D.W. Dickson, J.J. Augustine, and M.J. Basset. 1983. Reproduction of two races of Meloidogyne incognita in tomato plants grown at high temperature. J. Nematol. 15:640-641. Ayala, F.J., J.R. Powell, M.L. Tracey, C.A. Mourao, and S. Perez. 1972. Enzyme variability in the Drosophila willistoni group. IV. Genie variation in natural populations of Drosophila willistoni . Genetics 70:113-139. Boquet, D.J., C. Williams, and W. Birchfield. 1975. Resistance in soybeans to five Louisiana populations of the root-knot nematode. PI. Dis. Reptr. 59:197-200. Bush, G.L., and R.N. Huettel. 1972. Starch gel electrophoresis of tephritid proteins: A manual of techniques. Int. Biol. Program, Working Group on Fruit Flies. Population Genetics Project Phase 1. 56 pp. Calvo, B., and R. Lopez. 1980. Combate quimico de Meloidogyne incognita en dos cultivares de tabaco burley. Agron. Costarr. 4:175-182. Castro, J. A., and R. Lopez. 1981. Respuesta de dos cultivares de lechuga ( Lactuca sativa L.) a densidades crecientes de inoculo de Meloidogyne incognita (Kofoid y White) Chitwood. Agron. Costarr. 5:65-73. Carrillo, M. , and R. Lopez. 1979. Respuesta del tabaco estufado a la aplicacion de nematicidas. Nematropica 9:129-134. 114

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115 Chitwood, B.G. 1949. Root-knot nematodes-Part I. A revision of the genus Meloidogyne Goeldi, 1887. Proc. Helmin. Soc. Wash. 16:90-104. Christie, J.R. 1946. Host parasite relationships of the root-knot nematode, Heterodera marioni . II. Some effects of the host on the parasite. Phytopathology 36:340-352. Christie, J.R., and F.E. Albin. 1944. Host-parasite relationships of the root-knot nematode, Heterodera marioni . I. The question of races. Proc. Helmin. Soc. Wash. 11:31-37. Christie, J.R., and L. Havis. 1943. Relative susceptibility of certain peach stocks to races of the root-knot nematode. PI. Dis. Reptr. 32:510-514. Coetzee, V. 1956. Meloidogyne acronea , a new species of root-knot nematode. Nature 177:899-900. Dalmasso, A., and J.B. Berge. 1978. Molecular polymorphism and phylogenetic relationship in some Meloidogyne spp: Application to the taxonomy of Meloidogyne . J. Nematol. 10:323-332. Dalmasso, A., and J.B. Berge. 1979. Genetic approach to the taxonomy of Meloidogyne species, pp. 111-114. In F. Lamberti and C.E. Taylor (eds). Root-Knot Nematodes (Meloidogyne species). Systematics, Biology and Control. Academic Press, New York. Day, L.H., and W.P. Tufts. 1940. Further notes on nematode resistant rootstocks for deciduous fruit trees. Proc. Am. Soc. Hort. Sci. 37:327-329. Dickson, D.W., D. Huisingh, and J.N. Sasser. 1971. Dehydrogenases, acid and alkaline phosphatases, and esterases for chemotaxonomy of selected Meloidogyne , Ditylenchus , Heterodera and Aphelenchus spp. J. Nematol. 3:1-16. Dickson, D.W., and F.B. Struble. 1965. A sieving-staining technique for extraction of egg masses of Meloidogyne incognita from soil. Phytopathology 55:497 (Abstr.). Dropkin, V.H. 1953. Studies on the variability of anal plat patterns in pure lines of Meloidogyne spp. the root-knot nematode. Proc. Helmin. Soc. Wash. 20: 32-39. Eisenback, J.D., and H. Hirschmann. 1979. Morphological comparison of second stage juveniles of six populations of Meloidogyne hapla by SEM. J. Nematol. 11:5-16.

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116 Eisenback, J.D., and H. Hirschmann. 1980. Morphological comparison of Meloidogyne males by scanning electron microscopy. J. Nematol. 12:23-32. Eisenback, J.D., H. Hirschmann, J.N. Sasser, and A.C. Triantaphyllou. 1981. A guide to the four most common species of root-knot nematodes ( Meloidogyne species) with a pictorial key. A cooperative publication of the Departments of Plant Pathology and Genetics, North Carolina State University, and the United States Agency for International Development. North Carolina State Graphics. 48 p. Eisenback, J.D., H. Hirschmann, and A.C. Triantaphyllou. 1980. Morphological comparison of Meloidogyne female head structures, perineal patterns, and stylets. J. Nematol. 12:300-313. Esser, R.P., V.G. Perry, and A.L. Taylor. 1976. A diagnostic compendium of the genus Meloidogyne (Nematoda:Heteroderidae) . Proc. Helmin. Soc. Wash. 43:138-150. Figueroa, A. 1973. Estudio morfometrico y biologico sobre el nematodo cecidogeno del arroz Hypsoperine sp. (Nematoda:Heteroderidae) y pruebas de susceptibilidad al mismo de once variedades y una linea de arroz (Oryza sativa L.). Ing. Agr. Thesis. Universidad de Costa Rica. San Pedro de Montes de Oca, Costa Rica. 51 p. Fox, J. A., and L.I. Miller. 1973. Comparison of gall and egg-mass indices of two races of Meloidogyne incognita on ten differential hosts. Phytopathology 63:801 (Abstr.). Franklin, M.T. 1962. Preparation of posterior cuticular patterns of Meloidogyne spp. for identification. Nematologica 7:336-337. Giamalva, M.J., W.J. Martin, and T.P. Hernandez. 1963. Sweetpotato varietal reaction to species and races of root-knot nematodes. Phytopathology 53:1187-1189. Gonzalez, L. 1978a. Distribucion horizontal de algunos generos de nematodos f itoparasitos en terrenos agricolas de Costa Rica. Turrialba 28:67-69. Gonzalez, L. 1978b. Nematodos f itoparasitos asociados con la rizosfera de arroz y maiz en varias zonas agricolas de Costa Rica. Agron. Costarr. 2:171-173. Gonzalez, L. 1979. Nematodos f itoparasitos asociados con la cana de azucar en varias zonas de Costa Rica. Nematropica 9:32-35. Gonzalez, L. , and R. Lopez. 1980a. Evaluacion de nematicidas para el combate de Meloidogyne incognita en maiz. In Resumenes IV Congreso Agronomico Nacional y VII Congreso Latinoamericano de la Ciencia del Suelo. Colegio de Ingenieros Agronomos y Asociacion Costarricense de la Ciencia del Suelo. Heredia. p. 31.

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117 Gonzalez, L. , and R. Lopez. 1980b. Efecto de densidades de inoculo y caracteristicas del suelo sobre la patogenicidad de Meloidogyne incognita en lechuga. Agron. Costarr. 4:155-163. Goplen, B.P., E.H. Stanford, and M.W. Allen. 1959. Demonstration of physiological races within three root-knot nematode species attacking alfalfa. Phytopathology 49:653-656. Greet, D.N., and J. Firth. 1977. Influence of host plant on electrophoretic protein patterns of some round-cyst nematode females and use of larvae to obtain less ambiguous results. Nematologica 23:411-415. Hansen, E.L., and E.J. Buecher. 1970. Biochemical approach to systematic studies with axenic nematodes. J. Nematol. 2:1-6. Hidalgo, L., and R. Lopez. 1980a. Caracterizacion morfometrica de cuatro poblaciones de Meloidogyne incognita de Costa Rica. Turrialba 30:129-135. Hidalgo, L., and R. Lopez. 1980b. Susceptibilidad de diez cultivares de maiz ( Zea mays L.) a cuatro poblaciones de Meloidogyne incognita . Turrialba 30:316-322. Hirschmann, H. 1981. Morphological comparison of second-stage juveniles and males of members of the Meloidogyne incognita species complex using the scanning electron microscope. J. Nematol. 13:443 (Abstr.). Huettel, R.N., D.W. Dickson, and D.T. Kaplan. 1983. Biochemical identification of the two races of Radopholus similis by starch gel electrophoresis. J. Nematol. 15:338-344. Hussey, R.S. 1971. A technique for obtaining quantities of living Meloidogyne females. J. Nematol. 3:99-100. Hussey, R.S. 1979. Biochemical systematics of nematodes-A review. Helmin. Abstr., Series B, Plant Nematol. 48:141-148. Hussey, R.S. 1982. Molecular approaches to taxonomy of Heteroderoidea. pp 50-53. In R.D. Riggs (ed) . Fayetteville, Arkansas. Nematology in the Southern Region of the United States 276. Southern Cooperative Series Bulletin. Hussey, R.S., and K.R. Barker. 1973. A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. PI. Dis. Reptr. 57:1025-1028. Hussey, R.S., and J.N. Sasser. 1973. Peroxidase from Meloidogyne incognita . Physiol. Plant Pathol. 3:223-229. Hussey, R.S., J.N. Sasser, and D. Huisingh. 1972. Disc-electrophoretic studies of soluble proteins and enzymes of Meloidogyne incognita and M. arenaria . J. Nematol. 4:183-189.

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118 Incer, A., and R. Lopez. 1979. Evaluacion de practicas selectas para el combate integrado de Meloidogyne incognita en apio. Nematropica 9:140-146. Ishibashi, N. 1970. Variations of the electrophoretic protein patterns of Heteroderidae (Nematodea:Tylenchida) depending on the developmental stage of the nematode and on the growing conditions of the host plant. Appl. Ent. Zool. 5:23-32. Jepson, S.B. 1983. Meloidogyne kralli n. sp. (Nematoda:Meloidogynidae) a root-knot nematode parasitising sedge ( Carex acuta L. ) . Rev. Nematol. 6:239-245. Kirby, M.F. 1972. Florida root-knot nematodes characterized by morphology, host ranges, and enzymes and proteins separated with disc electrophoresis. M.Sc. Thesis. University of Florida. Gainesville, Florida. 80 p. Lopez, R. 1978. Nematodos f itoparasitos asociados al cultivo del tabaco ( Nicotiana tab a cum L.) en Costa Rica. Turrialba 28:279-282. Lopez, R. 1980a. Cambios estacionales de la distribucion espacial de nematodos en un plantio de higo. Nematropica 10:2 (Abstr.). Lopez, R. 1980b. Susceptibilidad comparativa de diez cultivares de frijol comun ( Phaseolus vulgaris L.) al ataque de Meloidogyne incognita . Agron. Costarr. 4:69-73. Lopez, R. 1980c. Determinacion de los nematodos f itoparasitos asociados al platano ( Musa acuminata x M. balbisiana , AAB) en Rio Frio. Agron. Costarr. 4:143-147. Lopez, R. 1981a. Distribucion espacial de nematodos del arroz despues de la cosecha en el sureste de Costa Rica. Agron. Costarr. 5:49-53. Lopez, R. 1981b. Observaciones sobre la distribucion espacial de Meloidogyne incognita despues de la cosecha en dos plantios de tabaco burley. Turrialba 31:11-14. Lopez, R., and J. Azofeifa. 1980. Nematodos f itoparasitos asociados a arboles frutales en algunos cantones de Alajuela. In Resumenes IV Congreso Agronomico Nacional y VII Congreso Latinoamericano de la Ciencia del Suelo. Colegio de Ingenieros Agronomos y Asociacion Costarricense de la Ciencia del Suelo. Heredia. p. 46. Lopez, R. , and J. Azofeifa. 1981. Reconocimiento de nematodos f itoparasitos asociados con hortalizas en Costa Rica. Agron. Costarr. 5:29-35. Lopez, R. , and D.W. Dickson. 1977. Morfometria y respuesta de hospedantes dif erenciales a tres problaciones de Meloidogyne incognita y una de M. javanica . Agron. Costarr. 1:119-127.

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119 Lopez, R. , and J. Fonseca. 1978. Combate quimico de nematodos fitoparasitos en tabaco de sol. Agron. Costarr. 2:157-162. Lopez, R. , and L. Salazar. 1978. Morfometria y algunos hospedantes de Meloidogyne hap la en la Cordillera Volcanica Central de Costa Rica. Agron. Costarr. 2:29-38. Lopez, R. , and L. Salazar. 1980. Influencia de la densidad inicial del inoculo sob re la patogenicidad de Meloidogyne incognita en pepino. In Resumenes IV Congreso Agronomico Nacional y VII Congreso Latinoamericano de la Ciencia del Suelo. Colegio de Ingenieros Agronomos y Asociacion Costarricense de la Ciencia del Suelo. Heredia. p. 32. Lopez, R. , L. Salazar, and J. Azofeifa. 1980. Observaciones sobre la distribucion espacial de nematodos asociados al cacao en Costa Rica. Nematropica 10:3-4 (Abstr.). Lordello, L.G.E, and A.P.L. Zamith. 1958. On the morphology of the coffee root-knot nematode, Meloidogyne exigua Goeldi, 1887. Proc. Helmin. Soc. Wash. 25:133-137. Martin, W.J., and W. Birchfield. 1973. Further observations of variability in Meloidogyne incognita on sweetpotatoes. PI. Dis. Reptr. 57:199. Mattey, J., and R. Lopez. 1978. Evaluacion de nematicidas y de metodos de aplicacion en el combate de nematodos fitoparasitos y en la producion y calidad de la lechuga. Turrialba 28:15-18. Nei, M. , and R. Chakraborty. 1973. Genetic distance and electrophoretic identity of proteins between taxa. Evolution 29:1-10. Netscher, C. 1978. Morphological and physiological variability of species of Meloidogyne in West Africa and implications for their control. Mededelingen Landbouwhogeschool. Wageningen, Nederland 78(3). 46 pp. Netscher, C, and D.P. Taylor. 1979. Physiological variation within the genus Meloidogyne and its implications on integrated control, pp 269-294. in F. Lamberti and C.E. Taylor (eds) . Root-Knot Nematodes (Meloidogyne species). Systematics, Biology and Control. Academic Press, New York. Olsen, K.L., and N.F. Thomas. 1954. Efectividad de dos fumigantes del suelo y dos insecticidas contra el nematodo de las agallas de las raices en tomates y okra. Turrialba 4:23-28. Padilla, C, and R. Lopez. 1979. Evaluacion de nematicidas granulados para el combate de Meloidogyne spp. en arveja ( Pisum sativum L.). Agron. Costarr. 3:89-95.

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120 Padilla, C, R. Lopez, and E. Vargas. 1980. Interacion entre Meloidogyne spp. y Fusarium oxysporum f . sp. pisi en arveja. Agron. Costarr. 4:55-60. Perlaza, F. , and R. Lopez. 1979. Endotoquia matricida en Meloidogyne incognita . Agron. Costarr. 3:45. Perlaza, F., R. Lopez, and E. Vargas. 1978. Efecto de la aplicacion combinada de nematicidas y fungicidas en el combate de Meloidogyne incognita , M. hap la y Alternaria sp. en lechuga. Fitopatologia 13:90-96. Perlaza, F. , R. Lopez, and E. Vargas. 1979. Combate quimico de Meloidogyne spp. y Alternaria sp. en zanahoria ( Daucus carota L.). Turrialba 29:263-267. Perry, V.G., and J. A. Zeikus. 1972. Host variations among populations of the Meloidogyne incognita group. J. Nematol. 4:231-232 (Abstr.). Pessoa, 0. 1973. Estudio evaluativo de cuatro nematicidas sistemicos en el tratamiento de rizomas de banano (Musa acuminata (AAA)) . Ing. Agr. Thesis. Universidad de Costa Rica. San Pedro de Montes de Oca, Costa Rica. 59 p. Priest, M.F., and C.J. Southards. 1971. Comparative morphology of sixteen isolates of Meloidogyne incognita . J. Nematol. 3:325-326 (Abstr.). Ramirez, A. 1971. Algunos aspectos sobre el control quimico de los nematodos del tomate. Ing. Agr. Thesis. Universidad de Costa Rica. San Pedro de Montes de Oca, Costa Rica. 39 p. Reynolds, H.W. 1949. Relative degree of infection of American-Egyptian and upland cotton by three populations of the root-knot nematode. PI. Dis. Reptr. 33:306-309. Riggs, R.D., and N.N. Winstead. 1959. Studies on resistance in tomato to root-knot nematodes and on the occurrence of pathogenic biotypes. Phytopathology 49:716-724. Rivera, R. , and R. Lopez. 1982. Evaluacion de nematicidas para el combate de Meloidogyne incognita en dos cultivares de Apium graveolens . Turrialba 32:67-73. Salas, L.A. 1975. La aparente tendencia a una distribucion geografica de algunas especies de nematodos f itoparasitos en Costa Rica. Fitopatologia 10:69 (Abstr.). Salas, L.A., and E. Echandi. 1961. Nematodos f itoparasitos en plantaciones de cafe de Costa Rica. Cafe 3:21-24. Salazar, L. 1980a. Variaciones morf ometricas y respuesta de nueve hospedantes dif erenciales a tres poblaciones de Meloidogyne javanica de Costa Rica. Turrialba 30:344-351.

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121 Salazar, L. 1980b. Distribucion vertical y horizontal de nematodos asociados a la cana de azucar en cinco zonas de Costa Rica. In Resumenes IV Congreso Agronomico Nacional y VII Congreso Latinoamericano de la Ciencia del Suelo. Colegio de Ingenieros Agronomos y Asociacion Costarricense de la Ciencia del Suelo. Heredia. p. 54. Salazar, L., and R. Lopez. 1980. Caracterizacion morfometrica y atnbito de hospedantes dif erenciales de diez poblaciones de Meloidogyne spp. de Costa Rica. Agron. Costarr. 4:21-31. Sancho, C.L. 1981. Patogenicidad de Meloidogyne sp. y determinacion de este y otros nematodos asociados al arroz ( Oryza sativa L.) en el Sureste de Costa Rica. Ing. Agr. Thesis. Universidad de Costa Rica. San Pedro de Montes de Oca, Costa Rica. 49 p. Sasser, J.N. 1954. Identification and host-parasite relationships of certain root-knot nematodes ( Meloidogyne spp.). Md. Agr. Exp. Sta. Bull. A-77. 30 pp. Sasser, J.N. 1966. Behavior of Meloidogyne spp. from various geographical locations on ten host differentials. Nematologica 12:97-98 (Abstr.). Sasser, J.N. 1972. Physiological variation in the genus Meloidogyne as determined by differential hosts. Eur. Mediterr. Plant Prot. Bull. 6:41-48. Sasser, J.N. 1979. Pathogenicity, host ranges, and variability in Meloidogyne species, pp 257-268. In F. Lamberti and C.E. Taylor (eds) . Root-Knot Nematodes ( Meloidogyne species). Systematics, Biology and Control. Academic Press, New York. Sasser, J.N. 1980. Root-knot nematodes: a global menace to crop production. PI. Dis. 64:36-41. Sasser, J.N., and C.C. Carter. 1982. Root-knot nematodes ( Meloidogyne spp.): Identification, morphological and physiological variation, host range, ecology and control, pp 21-32. In R.D. Riggs (ed) . Fayetteville, Arkansas. Nematology in the Southern Region of the United States 276. Southern Cooperative Series Bulletin. Sasser, J.N., and A.C. Triantaphyllou. 1977. Identification of Meloidogyne species and races. J. Nematol. 9:283 (Abstr.). Scotto la Massese, C. 1969. The principal plant nematodes of crops in the French West Indies, pp 164-183. In J.E. Peachey (ed) . London, England. Nematodes of Tropical Crops No. 40. Commonwealth Agricultural Bureaux. Technical Communication. Sherbakoff, CD. 1939. Root-knot nematodes on cotton and tomatoes in Tennessee. Phytopathology 29:751-752 (Abstr.). Siciliano, M.J., and C.R. Shaw. 1976. Separation and visualization of enzymes on gels, pp 185-209. In I. Smith (ed) . Chromatographic

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122 and Electrophoretic Techniques. Vol. 2. Zone Electrophoresis. William Heinemann Medical Books Publications, London, England. Sledge, E.B., and A.M. Golden. 1964. Hypsoperine graminis (Nematoda: Heteroderidae) , a new genus and species of plant-parasitic nematode. Proc. Helmin. Soc. Wash. 31:83-88. Southards, C.J., and M.F. Priest. 1973. Variation in pathogenicity of seventeen isolates of Meloidogyne incognita . J. Nematol. 5:63-67. Starr, J.L. 1979. Peroxidase isozymes from Meloidogyne spp. and their origin. J. Nematol. 11:1-5. Steiner, W.W.M. , and D.J. Joslyn. 1979. Electrophoretic techiques for the genetic study of mosquitoes. Mosquito News 39:35-64. Tarte, R. 1981. Informe sobre el progreso de la investigacion para el Proyecto Internacional Meloidogyne en Panama 1976-1978. pp 27-51. In R. Tarte (ed) . Ciudad de Panama, Panama. Memorias de la Segunda Conferencia Regional de Planeamiento del Proyecto Internacional Meloidogyne . Region I. International Meloidogyne Project . Taylor, A.L., and W.Q. Loegering. 1953. Nematodes associated with root lesions in abaca. Turrialba 3:8-13. Taylor, A.L., and J.N. S asser. 1978. Biology, Identification and Control of Root-Knot Nematodes (Meloidogyne species). A cooperative publication of the Department of Plant Pathology, North Carolina State University, and the United States Agency for International Development. Raleigh, North Carolina. North Carolina State Graphics. Ill p. Taylor, D.P., and C. Netscher. 1974. An improved technique for preparing perineal patterns of Meloidogyne spp. Nematologica 20:268-269. Triantaphyllou, A.C. 1981. Oogenesis and the chromosomes of the parthenogenetic root-knot nematode Meloidogyne incognita . J. Nematol. 13:95-104. Triantaphyllou, A.C. 1982. Cytogenetics and sexuality of root-knot and cyst nematodes, pp 71-76. In R.D. Riggs (ed) . Fayetteville , Arkansas. Nematology in the Southern Region of the United States 276. Southern Cooperative Series Bulletin. Triantaphyllou, A.C. , and J.N. S asser. 1960. Variation in perineal patterns and host specificity of Meloidogyne incognita . Phytopathology 50:724-735. Van der Laat, A. 1960. Ensayos para combatir la hernia radical del tomate, causada por nematodos del genero Meloidogyne , con fumigantes de suelo. Ing. Agr. Thesis. Universidad de Costa Rica. San Pedro de Montes de Oca, Costa Rica. 48 p.

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123 Viglierchio, D.R. 1978. Resistant host responses to ten California populations of Meloidogyne incognita . J. Nematol. 10:224-227. von Bulow, T. 1934. Informe anual 1933. Seccion de Patologia. Centro Nacional de Agricultura. Boletin No. 16. San Jose, Costa Rica, pp 56-62. von Bulow, T. 1935. Nota preliminar sobre la infestacion por nematodos de las raices del cafeto y de las ingas empleadas como sombra. Revista Instituto Defensa del Cafe de Costa Rica 3:29-33. von Bulow, T. 1936. Informe anual 1934. Departamento de Patologia. Centro Nacional de Agricultura. Boletin No. 18. San Jose, Costa Rica, pp 156-162. von Bulow, T. 1937. Informe anual 1935. Departamento de Patologia. Centro Nacional de Agricultura. Boletin No. 22. San Jose, Costa Rica, pp 74-78. Whitehead, A.G. 1968. Taxonomy of Meloidogyne (Nematodea:Heteroderidae) with descriptions of four new species. Trans. Zool. Soc. London 31:263-401.

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BIOGRAPHICAL SKETCH Roger Lopez Chaves, son of Roger Lopez C. and Francisca Chaves M. , was born November 8, 1945 in Atenas, Alajuela, Costa Rica. In 1963, and after completing his studies at the Liceo de San Jose, he entered the Universidad de Costa Rica. He graduated in August 1970 with the degree of Ingeniero Agronomo. From 1969 to 1973 he worked as a researcher in tobacco for a private company, and then joined the faculty at the Laboratorio de Nematologia, Facultad de Agronomla, Universidad de Costa Rica. In 1974 he entered graduate school at the University of Florida, where he obtained the Master of Science degree in 1976. After returning to Costa Rica he worked for five years before re-entering graduate school at the University of Florida to work on a Doctor of Philosophy degree. He is a member of the Society of Nematologists and the Organizacion de Nematologos de los Tropicos Americanos. He is married to Ana Isabel Chaverri M., and they have four children, Susana M. , Roberto E. , Jose F. and Juan P. Lopez Chaverri. 124

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. D. W. Dickson, Chairman Professor of Entomology and Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. G. C. Smart, Jr. Professor of Entomology and Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. R. E. Stall Professor of Plant Pathology

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. W. A. Dunn Associate Professor of Entomology and Nematology I certify that I have read this study and that in ray opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. R. Rich Associate Professor of Entomology and Nematology This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August 1984 Dean, College of Agriculture Dean for Graduate Studies and Research


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