Title: Rhizobium-related constraints to grain legume production in St. Kitts, West Indies /
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Title: Rhizobium-related constraints to grain legume production in St. Kitts, West Indies /
Physical Description: vii, 190 leaves : ill. ; 28 cm.
Language: English
Creator: Million, Jeff B ( Jeff Bacon ), 1957-
Publisher: s.n.
Publication Date: 1987
Copyright Date: 1987
 Subjects
Subject: Rhizobium   ( lcsh )
Legumes -- Saint Kitts and Nevis   ( lcsh )
Soil Science thesis Ph. D
Dissertations, Academic -- Soil Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1987.
Bibliography: Includes bibliographical references.
Statement of Responsibility: by Jeff B. Million.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00099322
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 001088027
oclc - 19299487
notis - AFH3400

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RHIZOBIUM-RELATED CONSTRAINTS TO GRAIN LEGUME
PRODUCTION IN ST. KITTS, WEST INDIES















By

JEFF B. MILLION


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

1987
















ACKNOWLEDGEMENTS


I wish to express my sincere appreciation to all those

who have provided support during this endeavor and who have

helped see it through to completion. Without these people

the project would not have been successful.

A giant thank you is extended to the chairman of the

supervisory committee, Dr. Jerry Sartain, for his unending

support and friendship. The work ethic he instilled in all

his graduate students will be greatly appreciated throughout

our careers.

I am deeply indebted to Dr. David Hubbell for

initiating this project and giving me the chance of a

lifetime to live and conduct overseas agricultural research

in St. Kitts. His optimism in times of discouragement will

always be remembered.

I would like to thank members of my supervisory

committee--Drs. William Blue, Kennth Buhr, and David Sylvia-

-for their guidance and support during the sometimes

uncertain phases of this project. The input of Dr. Kenneth

Quesenberry, as an original member of the supervisory

committee, is also greatly appreciated. Additionally, I

would like to thank the following persons: Drs. Kuell

Hinson and Alan Norden for furnishing peanut and soybean








seed, Mr. Edward Hopwood for aiding in soil characterization

work, Dr. Stewart Smith of the NITRAGIN CO. for supplying

inoculants, inoculant materials, and Rhizobium strains, Dr.

Fred Bliss of the University of Wisconsin for supplying bean

seed, and Dr. Adet Thomas of CARDI Jamaica for supplying

bean and cowpea seed.

A special thank you is forwarded to Mr. Austin Farier,

Ms. Jennifer Lowery, Dr. Ossi Liburd, Mr. Saga Browne, Mr.

Ashley Huggins, Mr. Jerome Thomas, and Mr. "Brotherman"

Huggins for making life and work easier for me and my family

in St. Kitts.

A final thank you goes to my wife, Amy, and our

daughter, Emily, who endured th campaign in St. Kitts and

sacrificed their time and energy in keeping me moving

forward towards completion.

























iii

















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS. . . . . . . .. . ii

ABSTRACT . . . . . . . . ... . . . vi

CHAPTERS

1 INTRODUCTION . . . . . . . . 1

2 LITERATURE REVIEW . . . . . . 9

Agriculture in St. Kitts . . . . . 9
Inoculation Strategies . . . . .. 14
Legume-Rhizobium Symbiosis . . . .. 21

3 FARM SURVEY . . . . . . . . 37

Soil Fertility of Farms in St. Kitts . . 37
Rhizobium Survey of Farms in St. Kitts . 53

4 FILTER-PRESS MUD AS AN ALTERNATIVE
INOCULANT CARRIER . . . . . .. 65

Introduction . . . . . . . . 65
Materials and Methods . . . . .. 66
Results and Discussion . . . . .. 70
Summary and Conclusions . . . . .. 74

5 BEAN FIELD INOCULATION TRIALS . . .. 76

Introduction . . . . . . . .. 76
Materials and Methods . . . . .. 77
Results and Discussion . . . . .. 85
Summary and Conclusions . . . . .. .108

6 SOYBEAN FIELD INOCULATION TRIALS . . .. .113

Introduction . . . . . . . .. 113
Materials and Methods . . . . .. 115
Results and Discussion . . . . .. 121
Summary and Conclusions . . . . .. .137











7 COWPEA AND PEANUT FIELD INOCULATION TRIALS . 141


Introduction . . . . . .
Materials and Methods . . .
Results and Discussion . . .
Summary and Conclusions . . .

8 SUMMARY AND CONCLUSIONS . . .

APPENDICES

A. SOIL FERTILITY DATA . . . . .

B. RHIZOBIUM CULTURE MEDIA AND SOLUTIONS

LITERATURE CITED . . . . . . .

BIOGRAPHICAL SKETCH . . . . . .














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

RHIZOBIUM-RELATED CONSTRAINTS TO THE
PRODUCTION OF GRAIN LEGUMES IN ST. KITTS, WEST INDIES

By

Jeff B. Million

December 1987


Chairman: Jerry B. Sartain
Major Department: Soil Science

Plans for the agricultural diversification in

sugarcane-dominated St. Kitts, an island-nation in the West

Indies, have included food legume production. Laboratory,

pot, and field studies were conducted to evaluate the

presence and effectiveness of indigenous rhizobia for

production of selected grain legumes in St. Kitts and to

develop a technology for local inoculant production

utilizing filter mud as the carrier material.

Uninoculated (-inoc) cowpeas (Vigna unguiculata L.

Walp) were nodulated in 34 out of 35 soils collected from

farms in St. Kitts while no nodules formed on uninoculated

soybeans (Glvcine max L. Merrill). For beans (Phaseolus

vulgaris L.), plant dry weight increases from inoculation

(+inoc) were observed in 11 out of the 30 soils in which a

response to N was observed. Ineffective strains were

suspected in seven out of those 11 soils.

vi








On-station and on-farm field inoculation trials were

conducted with cowpea and bean cultivars. No nodulation or

yield response to inoculation was observed in three trials

with cowpea, including one site which was recently taken out

of sugarcane production and which contained only 32 rhizobia

g-1 soil. Inoculation increased yields of two out of five

bean cultivars in an on-station trial in 1983. However, no

bean yield response was observed in an inoculation trial

conducted on the farm whose soil sample gave the greatest

increase (64%) from inoculation in the pot study. Under

high-yielding conditions at the research station, bean

yields were increased 20% (2380 vs 1970 kg ha-1) with the

application of 100 kg N ha-1; no yield response was observed

with inoculation.

Composted filter mud (FM) (390 g OM kg-l) was compared

to peat (870 g OM kg-1) as an alternative inoculant carrier.

Numbers of Rhizobium phaseoli and Bradyrhizobium iaponicum

were greater than 3 x 108 rhizobia g-1 in both materials

after 8 weeks of incubation. In the field, fine-sieved FM

inoculant seed-applied and coarse-sieved FM soil-applied

were as effective as peat inoculants in increasing soybean

yields. Inoculation increased soybean yields of cv. Santa

Rosa 24% and cv. UFV-1 12%, but had no significant effect on

cv. Jupiter; uninoculated controls yielded 2830, 3160, and

3440 kg ha-1, for the same three cultivars, respectively. A

local inoculant production program was recommended for

soybean production only.















CHAPTER 1
INTRODUCTION


The biotechnology of inoculating leguminous plants with

symbiotic N2-fixing Rhizobium bacteria has been

successfully exploited in many situations around the world.

By far the greatest and most successful use of Rhizobium

inoculants has been in soybean (Glycine max (L.) Merr.)

cultivation. The world-wide expansion of soybean

production has resulted in the development of inoculant

industries throughout the world (Burton, 1967; Thompson,

1980). This global expansion of inoculant production and

use was evident from observation of the countries

represented at the 1984 Inoculant Production Course offered

by the Nitrogen Fixation by Tropical Agricultural Legumes

(NifTAL). Countries represented included Brazil, Dominican

Republic, Egypt, Indonesia, Mexico, Morocco, Sudan, and

Thailand. Most of the representatives indicated that

soybean was the target legume in their respective

countries. However, large scale projects involving forage

legumes in Morocco and leuceana (Leuceana leucocephala) in

Sudan were primary factors for initiating inoculant

production in these two countries.

Research and experience have shown that maximum

benefits from symbiotic N2-fixation occur in soils with low










available N and under high-yielding conditions. Under

these conditions, soybean yield response to inoculation

averaged 3000 kg ha-1 or 70-80% of the total N accumulated

(Bezdicek et al., 1978). On the other hand, soybeans grown

in rotation with fertilized corn (Zea mays L.) generally

fix only 10-50% of their N (Harper, 1973). This phenomenon

is explained by the well-known fact that legumes have a

preference for soil inorganic N over symbiotic N so that

under high soil N conditions, nodulation and N2-fixation

are reduced. The majority of agricultural soils in the

tropics are low in N, especially under continuous cropping.

Whereas low soil N is a common occurrence, high-

yielding conditions necessary for the optimal expression of

symbiotic N2-fixation are of primary concern in the

tropics. Brathwaite (1982) found the following management

practices as most important for cowpea (Vigna unguiculata

(L.) Walp): pest control (144% increase over the control),

cultivar selection (77%), weed control (40%), rogue mozaics

(37%). and higher planting density (23%). Plant diseases

play a major limiting role in bean (Phaseolus vulgaris L.)

production (Graham, 1978). Nodulated legumes fix no more N

than required for yields defined by other environmental

(including soil N) and management factors (Singleton et

al., 1985). Therefore, the "Law of the Minimum" applies to

N2-fixation as well as other growth factors.

Cultivar selection plays a major role in maximizing

symbiotic N2-fixation in legumes. In general, later-










maturing cultivars tend to rely more on symbiotic N than

earlier-maturing cultivars (Graham and Rosas, 1977; Wynne

et al., 1982). Diversity of maturity types tends to be

greater with bean (bush versus climbing) and soybean

(photoperiodism) than with cowpea or peanut (Arachis

hypoqaea L.). Although maturity type is one important

criterion for cultivar selection, any characteristic which

may confer a yield advantage to one cultivar over another

should be considered. Important cultivar attributes may

include tolerance to drought, heat, soil acidity, diseases,

pests, etc. The relative importance of the above factors

will depend upon the location-specific characteristics of

the farm and the farmer's agronomic practices.

In order to assess whether or not nodulation and N2-

fixation are limiting to legume yields, inoculation trials

are conducted. In these experiments, nodulation and growth

of uninoculated plants are compared to those of plants

inoculated with known effective strainss. A N-fertilized

treatment is included to measure yields obtainable under

conditions where N is not limiting. In comparison with

inoculated and N-fertilized treatments, nodulation and

growth of uninoculated plants provide information relative

to the presence and effectiveness of indigenous rhizobia.

More specifically, several outcomes of an inoculation trial

are possible (Vincent, 1970; Date, 1982):

a) poor nodulation and growth of uninoculated plants
and no response to inoculation or N indicating
some other limiting factor such as nutrients,
water, weeds, etc.;








4

b) poor nodulation and growth of both inoculated and
uninoculated plants but good growth with N indica-
ting poor inoculant quality or application, or
that the strain was unsatisfactory;

c) poor nodulation and growth of uninoculated plants
but good nodulation and growth of inoculated
plants equal to N-fertilized plants indicating
that inoculation resulted in effective nodulation;

d) poor nodulation and growth of uninoculated plants
and good nodulation of inoculated plants but poor
growth relative to N-fertilized plants usually
indicating an ineffective inoculum strain;

e) nodulated control plants with poor growth and
nodulated inoculated plants with good growth
suggesting inoculum strain was competitive and
efficient;

f) nodulated control and inoculated plants with poor
growth relative to N-fertilized plants indicating
that either the inoculum strain was "out-
competed" by native strains or the inoculum strain
was ineffective (we need nodule strain
identification to distinguish between these two
interpretations);

g) well-nodulated control plants with good growth
indicating the presence of effective native
strains.


If results from the inoculation trials indicate an

inadequacy in local rhizobial populations and that this

inadequacy could be corrected through inoculant applica-

tion, a source of quality inoculant must be acquired. The

lack of readily available, fresh and high quality inocu-

lants is a major constraint to the adoption of the practice

of inoculation in many countries. Survival of rhizobia in

inoculants is quite variable and depends largely upon

conditions under which the inoculant has been stored. High

temperatures can result in a rapid decline in inoculant

quality during shipment and storage in the tropics (Somase-








5

garan et al., 1984; Roughley, 1968). Inconsistent

quality, coupled with the high cost of importing the

relatively inexpensive product, has resulted in the

generation of technologies which allow for local production

of quality inoculants utilizing locally available materials

(Somasegaran et al., 1982; Burton, 1984).

The culturing of Rhizobium in liquid broth prior to

addition to the carrier is a relatively simple process

requiring basic chemicals and labware. The most difficult

and time-consuming part of inoculant production is quality

control and maintenance of the Rhizobium strains from year

to year. This requires facilities for growing plants in a

controlled environment. The carrier material must be

locally available and compatible with the Rhizobium strains

to be used. Although peat has been the standard inoculant

carrier material, many other materials have been used with

varying degrees of success including compost, coal, coconut

coir, lignite, cellulose, bagasse, and charcoal (Graham,

1984). Research has shown that filter-press mud, a by-

product of sugarcane processing, has favorable properties

for growth and survival of rhizobia (Philpotts, 1976;

Khonje, 1983). If local production of inoculants is

warranted in St. Kitts, filter mud is a logical alternative

to peat. However, information is lacking relative to its

efficacy in the field.

St. Kitts is an independent island-nation situated in

the Leeward Island chain of the Lesser Antilles, West










Indies. Life in the island has been dominated agricul-

turally, politically, culturally and economically by the

sugarcane industry which has been in existence since the

1600s. However, due to declining world market prices for

sugar, renewed interest has been given to alternative

agricultural commodities. Despite the annual importation

of over 51,300 kg of dried grain legumes (St. Kitts

Statistical Division, data for 1981-1984), food legume

production in St. Kitts is scant and limited to peanut and

pigeonpea. The government-controlled National Agricultural

Corporation (NACO) has reduced peanut hectarage from 120 in

the period from 1975 to 1982 to less than 25 in 1983

(Caribbean Agricultural Research and Development Institute,

1985). Declining peanut production was due to the disrup-

tion and reduction of sugarcane plantings.

The lack of production of food grain legumes in St.

Kitts is due more to cultural and economic factors than to

agronomic factors. Due to the dominance of the sugarcane

industry, lands available to farmers are generally at

higher elevations, often on land inaccessible to farm

equipment. The local farmers tend to plant low-risk crops

such as root crops and banana which require little day-to-

day management. Very few farmers use improved agronomic

practices such as fertilization and pest control. These

traditional farmers must also deal with heavy crop damages

caused by monkeys and birds which thrive in the forest just

above their lands. The few enterprising farmers who have










adopted improved practices are planting vegetables, which

they are selling at a premium price at local markets.

Climatic and edaphic factors would appear to be

favorable for legume production. Rainy (fall) and dry

(spring) seasons exist although these are less noticeable

at higher (>350 m) elevations. Supplemental irrigation is

used when possible at the lower elevations for non-sugar-

cane crops. Excellent growing conditions are suggested by

the fact that St. Kitts once was named Liamigua or "fertile

earth." Soil characterizations of St. Kitts (CARDI, 1985;

Lang and Carroll, 1966; Walmsley and Forde, 1976) indicated

that sugarcane soils are well-drained loamy sands to loams,

have favorable pHs, and, except for N, generally contained

sufficient plant nutrient levels. No Rhizobium studies

have been reported for soils in St. Kitts.

The Caribbean Agricultural Research and Development

Institute (CARDI) is a regional agricultural organization

with a research station located in St. Kitts. The past

director of CARDI-St. Kitts was Dr. Laxman Singh who

initiated grain legume research in 1980 and 1981. During

that time, a collaborative research program between CARDI-

St. Kitts and the University of Florida was proposed by Dr.

David Hubbell of the Soil Science Department to conduct

Rhizobium studies parallel to on-going legume research

being carried out in St. Kitts by Dr. Singh. Support for

this research was ultimately funded through a Tropical

Agricultural Research grant administered by the United










States Department of Agriculture. Despite Dr. Singh's

leaving St. Kitts in 1982, the present research was initi-

ated in 1983.

The main objective of this research was to conduct

laboratory, pot, and field studies to assess potential

Rhizobium constraints to legume production in St. Kitts.

Soils were collected from farms around the island and

evaluated for potential fertility problems. Soils from

selected farms were also used to determine the presence or

absence of rhizobia infecting bean, cowpea, and soybean

plants. In addition, field inoculaton trials were con-

ducted at the CARDI Research Station and on a cooperating

farmer's land to evaluate the responses of different legume

species and cultivars to Rhizobium inoculation. Legume

species investigated in these trials included bean, cowpea,

peanut, and soybean. A small-scale inoculant production

technology utilizing filter mud as an alternative inoculant

carrier material was tested in the lab and in the field.













CHAPTER 2
LITERATURE REVIEW


Agriculture in St. Kitts

Much of the information presented in the following

description of St. Kitts and its agriculture was extracted

from two works. One was a soil and land use survey which

was conducted by Lang and Carroll (1966) of the University

of the West Indies. The other is a reconnaissance survey

of 120 farms throughout the island conducted in 1980 by

Singh and Lauckner (1981).


Description of the Island

St. Kitts is located in the Leeward Island group of

the northern Lesser Antilles at 170N and 620W. At its

greatest extent, St. Kitts is 32 km (19 mi) long and 10 km

(6 mi) wide. Of the 175 sq km (65 sq mi), approximately

40% (6,900 ha or 17,000 acres) has been devoted to

agriculture.

St. Kitts was built by volcanic activity initiated in

the Pleistocene epoch and continuing on until comparatively

recent times. Three more recent volcanic occurrences make

up the central ranges of the island where steep slopes and

tropical rainforest vegetation predominate from 500-1000 m

of elevation. The lower lands below 300 m are more

gradually sloping and these have been cultivated in








sugarcane over two hundred years. Much of the transition

zone between the sugarcane and the forest is cultivated by

small farmers, who can also be found working in erosion

gullies or "ghuts" which dissect upper sugarcane fields.

The climate of St. Kitts is very pleasant with annual

temperatures at lower elevations usually ranging from 18-

320C (65-900F); temperatures at higher elevations may drop

below 160C (600F). Temperatures are moderated by ocean

trade winds that blow constantly from the south-southeast.

Although rainfall does not vary much between the windward

and leeward sides of the island, wind blast causes some

crop damage and soils tend to dry out more rapidly on the

windward side.

Rainfall in St. Kitts closely follows altitude changes

with higher elevations receiving more rainfall than lower

elevations. Mean annual rainfall data collected over 18

years at sugarcane estates (NACO, 1983) is presented in

Table 2-1. Although some rain is normally received during

each month, highest rainfall occurs during July through

November with a rainy May month very common. However,

higher temperatures from March to August result in greater

evaporation so that the most favorable growing months are

from September through December. The dry season, which

extends from January to April, provides a low moisture (50-

75% relative humidity) environment conducive to har-

vesting and drying of crops.








Table 2-1.


Average rainfall and pan evaporation in St.
Kitts.


Month Rainfall Pan-evaporation Balance


- - - - cm (in.) - - - -

January 8.8 (3.5) 15.4 (6.1) -6.6 (2.6)

February 6.2 (2.4) 15.6 (6.2) -9.4 (3.8)

March 6.0 (2.4) 18.9 (7.4) -12.9 (5.0)

April 8.6 (3.4) 17.8 (7.0) -9.2 (3.6)

May 12.6 (5.0) 18.1 (7.1) -5.5 (2.1)

June 9.1 (3.6) 18.9 (7.4) -9.8 (3.8)

July 11.7 (4.6) 19.4 (7.6) -7.7 (3.0)

August 14.3 (5.6) 19.1 (7.5) -4.8 (1.9)

September 16.8 (6.6) 16.8 (6.6) 0.0 (0.0)

October 15.0 (5.9) 15.9 (6.3) +0.9 (0.4)

November 17.1 (6.8) 14.0 (5.5) +3.1 (1.3)

December 12.2 (4.8) 14.3 (5.6) -2.1 (0.8)


Total 138.2 (54.4) 196.6 (77.4) -58.4 (23.0)


at the NACO


Extracted from NACO (1983).
Thirty-year (1950-1980) average.
Thirteen-year (1970-1983) average measured
Agronomy Station (30 m elevation).








Soils in St. Kitts

The predominant agricultural soils of St. Kitts are

classified as Mollic Vitrandepts, although some Typic

Tropudalfs are found at higher elevations (Walmsley and

Forde, 1976). These loamy sand to loam-textured soils

ranged from pH 5.3 to 6.5, 24 to 30 g organic C kg-1, and

1.9 to 3.4 g total Kjeldahl N (TKN) kg-1. Cation exchange

capacity (CEC) ranged from 9.4 to 11.3 cmol(+) kg-1 soil

with base saturation ranging from 49 to 100%. Ammonium

acetate extractable K ranged from 160 to 420 mg

kg-1 while Olsen (0.5 M NaHCO3) P ranged from 10 to 39 mg

kg-1. A generalized description was given by Lang and

Carroll (1966) who also noted that the more highly

weathered soils at upper elevations had higher CEC values,

10-20 cmol(+) kg-1, variable but usually low exchangeable

K, and low P. They found certain allophanic soils at high

elevations to have up to 500 g OM kg-1.

Soil tests were conducted on 27 sugarcane fields to be

planted to a double crop of peanut (CARDI, 1985). Soil pH

ranged from 5.5 to 6.5, TKN from 1.2 to 2.4 g kg-1, and K

from 90 to 350 mg kg-1 (information on the extractant used

for K was not provided). Over 90% of the soils tested

sufficient in K according to the K sufficiency level of 100

mg kg-1 presented in the CARDI report. If a low critical

value of 1.7 g kg-1 is used for soil TKN (Walmsley and

Forde, 1976), almost 60% of the sugarcane soils tested

would be considered deficient in N. Truog P ranged from 24








to 412 mg kg-1 with over 80% testing above the low critical

value of 30 mg kg-1.

From the limited information available for soils of

St. Kitts, it appears that of all plant nutrients, N should

be most limiting, especially in non-sugarcane soils which

do not receive N fertilizer during the season. Phosphorus

was found to be adequate in sugarcane soils that receive

complete N-P-K fertilizer but the status of soil P in non-

sugarcane fields should be less.


Description of Farming in St. Kitts

Some very interesting results came out of a socio-

economic survey conducted by Singh and Lauckner (1981). Of

the 900 small farmers in the island, 120 were selected for

the survey. Over one-third of the farmers worked less than

0.5 ha; only five out of 120 farmers had farms over 2 ha.

The small scale of operations is evident from the finding

that only six of 120 farmers used back-pack sprayers, while

none had tractors or irrigation equipment.

Almost 90% of the farms were on either gradually

sloping or almost flat land. Rainfall was estimated to be

between 102 and 152 cm (40 and 60 in) per year for 63% of

the farms; 35% were estimated to receive over 152 cm (60

in).

Crop production was dominated by root crops (e.g.

sweet potatoes (Ipomoea batatis Lam.), yams (Discorea sp.),

tannia, dasheen, and eddoes) and bananas. The most popular

vegetables grown were cabbage (Brassica oleracea capitata








oleracea capitata L.) (28% of farmers), carrots (Daucus

sp.) (27%), tomato (Lycopersicon esculentum Mill.) (21%)

and pumpkin (Cucurbita sp.) (21%). Of the grain legumes,

only peanut (Arachis hypoqaea L.) (10%) and pigeonpea

(Calanus cajan L.) (13%) were grown by the farmers. Peanut

plantings were from July to September and were harvested in

January.

Only 14% of farmers were under the age of 40. Over 75%

could read and write. One-fifth of the farmers were full-

time farmers, the majority holding jobs as laborers in the

seasonal sugarcane operations. Over 60% of the farmers had

family incomes less than $EC 2,500 ($EC 1.00 = $US 0.38);

none divulged incomes greater than $EC 10,000. The above

descriptions indicate that farming outside of sugarcane

production is rudimentary and on a subsistence level.

The absence of bean and cowpea production is surprising

considering that during 1981 to 1984 St. Kitts imported an

average 38,100 kg of kidney bean at an average price of EC$

2.49 per kg; import data for pink beans and blackeye

cowpeas were not provided separately but were probably as

great (data obtained from the Statistics Division of the

Planning Unit, St. Kitts Government). Average total grain

legume imports averaged 51,300 kg at a price of EC$

117,000.


Inoculation Strategies

Rhizobium inoculation describes the practice of

artificially introducing Rhizobium bacteria in high numbers








either directly to the legume seed or indirectly to the

soil into which the seed is planted. Many different

inoculant products and inoculation methods have been

developed and successfully utilized by farmers. Several

important points are now reviewed with respect to inoculant

use in St. Kitts.


Inoculants and Inoculant Carriers

The history of inoculants has taken us from rhizobia-

populated soil, to pure broth or agar cultures, and finally

to peat which has been impregnated with rhizobia (Date and

Roughley, 1977; Burton, 1967). Due to ease of handling and

time saved by avoiding seed inoculation, liquid inoculum

has gained in popularity in mechanized agriculture in the

U.S. (Burton, 1979). According to Burton, a carrier should

have several attributes, including: (1) support rhizobial

growth (non-toxic), (2) be highly absorptive, (3) be easily

processed (milled and sterilized), (4) adhere well to

seeds, and (5) be in readily available supply at low cost.

In addition to having the above capabilities, peat enhances

survival of rhizobia on inoculated seed (Vincent, 1970;

Burton and Curley, 1965). Peat has proven successful in

major inoculant production industries in the U.S. and

Australia and is the standard material against which all

alternative carriers are tested (Strijdom and Deschodt,

1976).

Alternative carrier materials have been successfully

used where peat was not available or was available only at








uneconomical costs. Common to most carriers is some form

of organic material which serves as a carbon source for the

bacteria. In Sudan, Mukhtar and Abu Naib (1987) used what

they termed "Nile silt" which consisted of 90 g Nile silt

soil, 5 g charcoal, 4 g bagasse, and 1 g sucrose. Faizah

et al. (1980) recommended a compost made from 8 g coir-

dust, 25 g loamy sand soil, 5 g CaC03, and 60 ml H20.

Lignite, which can be improved with a soybean meal

amendment, is a carrier material used in India (Kandasamy

and Prasad, 1971). A cellulose powder collected from a

cotton factory proved adequate, but was not very promising

due to rapid moisture loss during initial storage

(Pugashetti et al., 1971). Bagasse ground to pass a 275

mesh (0.053 mm) screen was adequate up to 100 days

(Leiderman, 1971). Bagasillo, a fine dust which separates

from the bagasse in the sugarcane mills, is used in

Zimbabwe (Ryder and Grant, 1983). Due to its very high

water-holding capacity (WHC), bagasillo absorbs twelve to

thirteen times its own weight. Bagasillo was brought to 40%

WHC with a salt solution and autoclaved for 2 hr in high-

density polyethylene bags. The final product after

addition of the broth culture contains 5.9 g bagasillo and

47.9 mL solution of which 2.5 mL are broth culture. They

claimed over 1010 rhizobia g-1 inoculant. Graham (personal

communication) suggested washing fresh bagasse and filter

mud prior to use in order to remove excess sucrose,

especially for slow-growing rhizobia. Coal-based inocu-

lants compared favorably with peat (NITRAGIN) and were








better than lignite-based inoculants for soybean nodulation

in the field (Dube et al., 1974). Mixtures of coal with

vermiculite, corn meal, sucrose and yeast extract did not

improve the survival of a bean strain (Paczkowski and

Berryhill, 1979). Charcoal and vermiculite were successful

carriers; ground peanut hulls and corn cobs were not

(Sparrow and Ham, 1983). Graham (1982) tested soil-

charcoal (99:1) and bagasse-charcoal (99:1) mixtures.

Survival was poor for both materials as populations dropped

from 109 to 107 after only 9 days.

Filter-press mud has been found to support high

populations of rhizobia. Philpotts (1976) studied the

survival of a fast-growing clover strain and a slow-growing

cowpea strain in filter mud. Filter mud was either air-

dried or oven-dried and ground to pass a 0.75 mm screen.

Both non-sterile and pre-sterilized (autoclaved) filter mud

carriers were inoculated with broth cultures of the

Rhizobium strains and survival was monitored over an eight

week period. Both non-sterile and autoclaved material

contained more than 108 rhizobia g-1 inoculant after 8

weeks. In one case, survival of the fast-growing clover

strain was reduced in the unsterilized filter mud. Oven-

drying was detrimental to survival but this effect was

diminished if the dried filter mud was stored for 3 months

before use.

In Malawi, inoculant production switched from a soil-

rice husk carrier to filter mud (Khonje, 1983). For their

purposes, the filter mud was air-dried and ground to pass a








100 mesh (0.15 mm) sieve. This filter mud had a pH of 8.3

(0.01 M CaC12) and contained 350 g OM kg-1. The material

had 62% moisture at pF=3.9 (equivalent to 0.28 bar or

28kPa) considered to be the moisture tension at which

optimum growth and survival of rhizobia occurs (Date and

Roughley, 1977). When inoculated with a fast-growing

leuceana strain and a slow-growing cowpea strain, viable

cell counts were greater than 109 rhizobia g-1 after 8

weeks. Both strains increased over ten-fold during the

first 2 weeks indicating that the filter mud has favorable

properties for growth of the rhizobia.

Although these studies have shown that filter mud is

an acceptable medium for growth and survival of rhizobia,

no field studies which evaluate the efficacy of filter mud

in the field were found in the literature.

Quality standards have been set by inoculant manufac-

turers and regulating agencies. In Australia, inoculum

broth and fresh peat inoculants are tested by the

Australian Inoculants Research and Control Service (AIRCS)

before products are sold to retail outlets (AIRCS, 1984).

A batch of inoculant passes inspection if it meets the

following criteria: (1) it contains 109 rhizobia g-1 moist

peat, (2) its cells are Gram-negative and sereologically

correct, (3) there is no contamination on 10-6 dilution

plates, (4) it forms nodules on test host at both the 107

and 108 dilutions, and (5) the inoculant contains greater

than 45% moisture. Inoculants sold at retail outlets must

have one-tenth the standard for fresh inoculants, i.e. 108








rhizobia g-1 moist peat. Pre-inoculated seed must have

more than 103 rhizobia per seed. In the U.S., simple grow-

out tests are used to assess inoculant quality. These

trials are considerably less stringent than the quality

control procedures of Australia (Burton, 1967).


Inoculation Methods

Inoculants can be applied either directly to the seed

or indirectly to the soil. Seed inoculation has been a

successful practice in most situations, especially with the

advent of high quality peat inoculants and seed-coating

adhesives. However, in certain cases, soil inoculation has

provided better results by either allowing for a physical

separation between rhizobia and seed protectants (Graham et

al., 1980) or by allowing for greater inoculum rates.

Scudder (1975) found seed inoculation to be unsatis-

factory in the hot, sandy soils of Florida, especially for

the first soybean planting. Application of granular

inoculant (6.7 x 108 rhizobia g-1) at 5.6 kg ha-1 or liquid

inoculant (9.7 x 108 rhizobia ml-l) at 3.9 L ha-1 resulted

in a tenfold increase (8.7 x 106 vs 8.3 x 105) in the

number of rhizobia per seed over standard seed inoculation.

Soil application of the granular and liquid inoculants

increased plant color ratings, nodulation, and seed yield

of two soybean cultivars; no differences were observed

between the liquid and granular inoculants.

Soil-applied inoculants can be advantageous in soils

with either low or high populations of indigenous rhizobia.








Bezdicek et al. (1978) found granular inoculants to give

greater soybean yields in a rhizobia-free soil even though

peat-based seed inoculation provided greater inoculum

rates. In a Bradyhizobium japonicum-free soil in Puerto

Rico, soybean cv. Jupiter was sown with liquid inoculant at

inoculum rates up to 9.6 logl0 rhizobia cm-1 row. Inoculum

rates greater than 5.6 logl0 rhizobia cm-1 row were

required for the formation of more than three nodules per

plant at 53 days. Seed yields were unaffected by inocula-

tion or 100 kg N ha-1 indicating high soil N reserves. No

soil N data were provided.

High inoculum rates afforded by granular and liquid

inoculants can increase the number of nodules formed by

inoculum strains in rhizobia-populated soils but overall

nodule number and yields are often unaffected. Thus, in

soils with more than 103 rhizobia g-1 soil, nodulation was

not increased by inoculation (Weaver and Frederick, 1974).

Weaver and Frederick found that inoculum rates greater than

1000 times the soil populations were required for greater

than 50% recovery of inoculant strains. However, in these

midwestern soils, soil inorganic N was high (50-100 kg

ha-1), nodule counts were low (1-25 nodules plant-1), and

no yield response to inoculation was obtained. Similar

results were obtained by Boonkerd et al. (1978) who

observed no nodulation or yield response of soybean to 10

and 100 times normal inoculum rates in rhizobia-populated

soils in Maryland. However, percent recovery of inoculum

strains was increased up to 50% at the highest inoculum








rates (4.4 x 1012 cm-1 row). Soil-applied inoculants at

inoculum rates greater than 105 rhizobia cm-1 row were

needed to achieve the greatest increase in nodulation and

seed yield of beans in a low N soil (Sparrow and Ham,

1983).

An alternative to the use of granular or liquid

concentrate inoculants is to make a slurry with powdered

inoculant and spray or gravity feed it into the furrow

during the seeding operation. Good agitation in the tank,

spray pressures below 170 kPa, and double filtration

systems were needed for successful field operation

(Brockwell and Gault, 1982). Suspension of rhizobia in

water did not decrease rhizobia viability below 107

rhizobia per mL-1 and had little effect on subsequent

effectiveness (Crist et al., 1984). In order to achieve

successful inoculation in the hot, dry and sandy soils of

Israel, high inoculum rates are met by using granular

inoculants or by spraying a peat-water suspension into the

furrow (Okon et al., 1979; Schiffman and Alper, 1968). A

system which can deliver a water-suspension of solid

inoculant may be a successful alternative to using granular

inoculant which requires more material and sophisticated

mechanization to deliver the low rates. In addition, the

water suspension may provide more favorable soil conditions

for the introduced rhizobia.








Lequme-Rhizobium Symbiosis

A legume can derive most of its N requirement through

symbiotic relationships with N2-fixing rhizobia. Dart and

Krantz (1977) reported that groundnut can fix up to 240 kg

N ha-1 or 80% of its total N accumulation. Soybean can

derive almost all its N requirement from N2-fixation under

low soil N conditions commonly encountered in the sandy

soils of Florida, but high inoculum rates are important,

especially in the first year of production (Hinson, 1974;

Scudder, 1975). Soybean fixed 80% of its N requirement

which was over 300 kg N ha-1 in a N-deficient soil

(Bedzicek et al., 1978). Westermann et al. (1981) found

bean cultivars grown in Idaho fixed an average 90 kg ha-1

or 40-50% of the total N found in bean plants at maturity.

Nitrogen fixation by field beans is quite variable with

later-maturing, indeterminate types depending more upon N2-

fixation than early-maturing determinate types (Graham,

1981). Symbiotic N2-fixation can supply 90% of the N

requirement of high-yielding cowpeas (Eaglesham et al.,

1977).

Many factors affect the amount of N fixed by the

legume-Rhizobium symbiosis. Genetic factors of both the

host legume macrosymbiont and the bacteria microsymbiont

determine the potential for effective N2-fixation while

environmental factors dictate the actual amount of N fixed

by the legume plant under field conditions. While strains

vary in their capacity to nodulate and effectively fix N

(Halliday, 1983), excellent inoculant strains are now








available for most legumes. It is not surprising, there-

fore, that host factors play a major role in effecting

changes in biological N fixation (BNF) of nodulated legumes

(Graham, 1982).

In order to optimize BNF in legume production, two

central questions must be addressed. The first question

regards whether or not Rhizobium inoculation is needed, and

if so, how can it best be accomplished. The second question

concerns whether or not high-yielding and N-limiting

conditions exist. High-yield conditions will occur with

proper cultivar selection, adequate soil fertility, good

disease and pest control, adequate moisture and tempera-

ture, plant populations, etc. Because answers to these

questions depend largely on the legume species, the

following discussion of Rhizobium inoculation and legume

agronomy is divided among several species: beans, soybeans,

cowpea, and peanut.


Beans (Phaseolus vulgaris)

The common bean is an annual pulse grown for fresh,

mature pods or dry food grain. There is a wide range of

seed types (color, shape, and size), disease resistance,

maturity, and growth habits. The Centro Internacional de

Agriculture Tropical (CIAT) in Colombia maintains a large

bean germplasm collection and coordinates much of the

international research on beans (CIAT, 1981).

Beans are generally considered to be poor N2-fixers

relative to other major grain and forage legumes. Hardy et








al. (1968) reported that the specific nodule activity (SNA

= mol C2H4 g1- nodule hour-1) of bean was less than half

that of soybean indicating that genetic factors are

limiting N2-fixation. On the other hand, Graham and Rosas

(1977) compared SNA of 20 bean cultivars and found maximum

SNA of 130-200 mol C2H4 g-1 nodule hr-1 which was greater

than 103 mol C2H4 g-1 nodule hr-1 observed for a soybean

cultivar. Piha and Munns (1987) compared the N fixation

capacity of nine bean cultivars with two cultivars each of

cowpea and soybean. All matured at approximately the same

time thereby eliminating differences in days to maturity.

Soybeans fixed more N (non-nodulating isoline difference

method) than beans. Total N fixed by beans (7-123 kg ha-1)

was considerably less than by cowpea (185-206 kg ha-1) or

by soybean (179-199 kg ha-1). Shoot N at 50 days was

increased with N fertilizer for beans but much less so for

cowpea and soybean. The N-accumulation rate during pod-

filling under N fertilization was less for bean indicating

that N2-fixation in bean may be limited by the plant's

capacity to accumulate N. They concluded that N2-fixation

plays a greater role in the later stages of bean growth

while nitrate assimilation is more important early on in

development. On the other hand cowpea, which flowered at

53 days, had a longer vegetative period to establish the

N2-fixing system than beans which flowered earlier and

depended on N2-fixation at a time when pods were more

successfully competing for limited photosynthate.






25

Some of the confusion surrounding N2-fixation in beans

is due to the wide range in N2-fixation exhibited by

different bean cultivars. Graham (1981) reported that the

acetylene reduction activities (ARA) of indeterminate

climbing cultivars were considerably greater than those of

determinate bush cultivars and were comparable to the ARA

of other legumes. Graham discussed three factors

contributing to this increased capacity for N2-fixation in

climbing beans: increased partitioning of non-structural

carbohydrate to the nodules, decreased uptake of soil N,

and a longer vegetative period. A longer vegetative period

for cv. Aurora resulted in an average seasonal fixation

increase of 41% (119 vs 84 kg N ha-1) over cv. Kenwood

which matured 8 days earlier (Rennie and Kemp, 1984).

Nitrogen fixation accounted for over 60% of N accumulated

by these cultivars. Hardy and Havelka (1976) argued that

by extending the vegetative period 9 days, seasonal N2-

fixation could double.

During early growth, leaf N assimilation was greater

for bush beans than for climbing varieties (Graham and

Rosas, 1977) indicating that bush beans are more responsive

to fertilizer N. Rennie and Kemp (1983) reported that N2-

fixation by cv. Redkloud was unaffected by N at 40 kg ha-1

while N2-fixation of cv. Limelight was reduced 60%.

Differential effects of N on cultivars were also found by

Westermann et al. (1981) who showed that N fertilization

decreased N2-fixation (C2H4) 90% for two cultivars and only

50% for two others. Results of Rennie and Kemp (1983)








showed that percent plant N derived from N2-fixation was

less for three bush bean cultivars than for two semi-vining

cultivars. They also found that ARA was a poor predictor

of total N fixed. Hence, pink bean cvs. Sutter Pink and

Viva fixed the same amount of N (15N method) even though

Viva exhibited an eightfold increase in ARA.

Seed yields of bean cv. Canadian Wonder increased up

to the highest addition of 200 kg N ha-1 in Malawi (Edje et

al., 1975). Percent seed N was increased with increasing N

rate. Yields were more highly correlated with pods per

plant than seed per pod. Despite increasing yields, 200 kg

N ha-1 had little effect on seed N except for bean cv. Red

Kidney for which seed N increased from 32.8 to 36.4 g N

kg-I seed (Piha and Munns, 1987). Westermann et al. (1981)

suggested that low N (less than 40 kg ha-1) fertilizer

rates should increase bean yields in soils testing less

than 50 kg available N ha-1.

Attewell and Bliss (1985) initiated a breeding program

to incorporate higher N2-fixation traits into lower N2-

fixing, standard commercial cultivars. Puebla 152 was the

donor parent while widely adapted cvs. Porillo Sintetico

(21 series) and Sanilac (24-series) were used as recurrent

parents. Four progeny selections from the 21 series fixed

25-40% more N than their recurrent parent, Porillo Sinte-

tico; selections from the 24 series fixed 150-670% more

than the recurrent parent Sanilac. Line 24-17, which fixed

670% more N than its parent, Sanilac, and line 21-58, which

fixed 28% more than its parent, Porillo Sintetico, were








included in a bean inoculation trial in St. Kitts to be

described later. Despite increased N2-fixation, none of

the progenies matched the fixation of the donor Puebla 152.

The growth habit of 24-17 was semi-vining while its

recurrent parent, Sanilac, was non-vining. In a follow-up

study, Dubois et al. (1985) found cultivars varied from 10-

33% in the amount of N fixed by the R3 stage. Line 24-17

exhibited greater N2-fixation than the recurrent parent

while retaining greater partitioning of fixed N to the

seeds than the high N2-fixing donor Puebla 152.

In east Africa, Keya et al. (1982) surveyed producing

regions for bean rhizobia. They tested soils by making

dilutions and inoculating aseptically-grown beans in pots.

Only six of the 68 soils tested were devoid of bean

rhizobia. Growth responses from soil inoculation ranged

from 53-625%. Graham et al. (1982) reported positive bean

yield responses to inoculation ranging from 39-61% in five

of 12 sites in Colombia. Sartain et al. (1982) reported

variable effects of N and inoculation on yields of red and

black beans in El Salvador. At one site, higher yielding

black bean cvs. Porillo 70 and S-184N responded to N while

lower yielding red bean cvs. Rojo de Seda and Nahuizalco

Rojo did not. Inoculation with a CIAT inoculant increased

yield of Porillo 70 only. At another lower-yielding site,

inoculation, but not N fertilization, increased bean

yields. At a third site, inoculation resulted in a de-

crease, increase, or no effect depending upon the cultivar,

inoculant strains, and inoculant form. These results








demonstrate the need for multilocational and multivarietal

trials when attempting to ascertain the potential benefits

of inoculation in a given region.


Soybean (Glycine Max)

Soybean is a warm season, annual grain legume indige-

nous to southeast Asia. Breeding of improved varieties has

resulted in cultivars adapted to a wide range of climates

(INTSOY, 1982). The seed is very high in protein (40%) and

oil (20%) making it a major industrial crop. The increase

in world production in recent years is supported by the

crop's high protein productivity (9.1 kg ha-1) relative to

other legumes, e.g. peanut (2.7 kg ha-1), and cowpea (3.3

kg ha-1), root crops, e.g. sweet potato (Beta vulqaris)

(1.4 kg ha-1), and cereals, e.g. corn (1.6 kg ha-1) and

rice (Oryza sativa L.) (1.9 kg ha-1) (Moomaw et al., 1977).

In most countries where soybean has recently been

introduced, improved soybean cultivars respond dramatically

to inoculation with effective strains of Rhizobium.

Positive yield responses to inoculation are reported from

Nigeria (Kang, 1975), Israel (Okon et al., 1979), Ghana,

Sierra Leone, Cameroon, Malagasy Republic, Zaire, Kenya

(Ayanaba, 1977), and Sudan (Khalifa, 1987). Sundara Rao

(1971) reported inoculation responses at 7 of 7 sites in

India. Due to the consistent response of introduced

soybeans to Rhizobium inoculation, a large inoculant

production industry has developed in Brazil where soybean

production has expanded from 80,000 ha in 1955 to 8 million








ha in 1975 (Jardin Freire, 1977) and over 65% of soybeans

planted are inoculated (Graham and Halliday, 1977).

Although yield increases have been reported for N-

fertilized soybeans (Khalifa, 1987; Sorensen and Penas,

1978; Bhangoo and Albritton, 1972), N fertilization is

generally not recommended except under very low soil N

conditions at which time starter applications may be

beneficial (Hinson, 1974). Thus, Beard and Hoover (1971)

found that even though early N-deficiency symptoms were

observed for unfertilized soybeans, yields were similar to

those of soybeans that had received N. Chesney (1973)

reported no effect of starter N up to 44 kg ha-1 on soybean

yield during four seasons. Results from the International

Soybean Variety Evaluation Experiments (ISVEX) coordinated

by the International Soybean Program (INTSOY) indicated

that starter N applications which averaged 20 kg ha-1 had

very little effect on soybean yield while nodule weight per

plant was highly correlated with yield (Whigham et al.,

1978).

Soybean has traditionally been viewed as requiring

specific B. japonicum strains for nodulaton and N2-fixa-

tion. Recent investigations indicate some cultivars are

more "promiscuous" than others. In Trinidad, uninoculated

soybean cv. Jupiter was effectively nodulated by indigenous

rhizobia (Awai, 1980). Inoculation with either a local

isolate or imported strains increased yields. Whereas

early nodulation was enhanced by inoculation, nodulation by

indigenous strains was prominent late in the season,








indicating low populations. In Nigeria, Jupiter nodulated

with native cowpea rhizobia only to a very limited extent

(IITA, 1979). United States cvs. Jupiter and Bossier were

effectively nodulated with slow-growing B. japonicum but

not with fast-growing B. laponicum or cowpea rhizobia

(Eaglesham, 1985). Indigenous rhizobia nodulated better

with Indonesian soybean cv. Orba than U.S. cultivars (Rao

et al., 1985). Research is currently being conducted to

incorporate the higher agronomic potential of U.S. culti-

vars into the more promiscuously nodulating Asian types

(Chowdhury, 1977; Nangju, 1980; Ranga Rao et al., 1982).

The greatest benefits of BNF will be realized under

high yielding conditions. Since most commercial soybean

varieties are photoperiod sensitive, cultivar selection and

planting date are important decisions to be made. Hinson

(1974) discusses important plant and environmental traits

for soybeans in low-level tropical locations including

(1) have a determinate growth habit,

(2) be planted 20 to 50 days prior to maximum
daylength (late May or early June in north
latitudes),

(3) have adequate moisture except after maturity is
reached,

(4) require 42 to 50 days to first flower,

(5) require 120 days to maturity,

(6) form 15 nodes and be 85 to 100 cm in stem length,

(7) form a closed canopy during pod-filling.

Soybean cv. Jupiter, which has been adopted as a standard

in international trials, flowered 47 and 37 days after






31

planting (DAP) when planted 170N latitude (same latitude as

St. Kitts) in April and August, respectively (INTSOY,

1983). Days to maturity for the same site and planting

dates were 165 and 101, respectively. Soybean cv. UFV-1,

also a late-maturing cultivar from the IX Maturity Group,

flowered a week earlier than Jupiter and matured 4 days

earlier. In Puerto Rico (180N), highest yields are

obtained for plantings in June, July, and August (Samuels,

1969).

Although planting in June under long daylengths would

result in the flowering and maturity periods recommended by

Hinson above, rainfall patterns should be a more important

criterion in St. Kitts where rainfall is highest in October

and November. Low and inconsistent rains are received in

June and July (Table 2-1). Therefore, planting as soon as

the rains start in the fall would probably be the most

favorable planting strategy in St. Kitts. An early fall

planting should result in soybean maturing during the

normally dry months of December and January.


Cowpea (Viqna Unquiculata)

Cowpea is the most popular grain legume in Africa.

There, semi-vining types are intercropped with sorghum or

millet while more determinate, day-neutral types are

intercropped with root crops, maize, or sole-cropped in the

more humid areas (Dart and Krantz, 1977). Blackeye cowpeas

are also popular in the Caribbean region where farmers

produce 1550 metric tons of cowpea while consumers demand








5450 metric tons (Ferguson and Jallow, 1984). Cowpea have

fewer disease problems than bean but are more affected by

insects, especially pod-sucking insects.

References in the literature describing growth and

yield response of cowpea to inoculation are few. Sellschop

(1962) reported that cowpea do not respond to inoculation

in Africa. Ayanaba and Nangju (1973) also reported limited

or no yield response of cowpea, lima bean, and pigeonpea to

inoculation in Africa. Yields of cowpea cv. Los Banos Bush

Sitao No. 1 were unaffected by inoculation or N fertilizer

applications up to 120 kg ha-1 in Trinidad (Graham and

Scott, 1984). Inoculation and N (40 kg ha-1) increased

shoot weight and total N at 40 DAP but yields were

unaffected. Although yield responses to inoculation have

been inconsistent in multi-location trials in India, Hegde

(1977) reports 11 of 21 cultivars responded to inoculation

during five seasons of testing at several sites.

Varietal differences in N2-fixation are reported for

cowpea. Graham and Scott (1983) evaluated 12 cowpea

cultivars in Trinidad and found high correlations between

total N and shoot weight, total N and nodule weight, and

total N and seed yield. The authors contend that the wide

differences in yield were due to genetic differences in N2-

fixing potential. However, it is unlikely that the five-

fold differences observed in their trial can be accounted

for by differences in N2-fixation activity alone. It is

more likely that the cultivar's yield potential affected








N2-fixation activity more than N2-fixation activity

affected yield.

Zary et al. (1978) found a wide range in ARA among 100

cowpea genotypes. They found ARA assayed in greenhouse

screenings to predict well the ARA of cowpeas in the field.

However, dry matter production in the field was not well

correlated with ARA or nodule weight per plant.

More than 400 lines of cowpea were evaluated under -N

and +N fertilizer treatments at three locations in Nigeria

and the Republic of Niger (Ahmad et al., 1981). Nodulation

and relative effectiveness ratings (-N shoot wt/ +N shoot

wt) differed widely between cowpea lines and between sites.

Effectiveness ratings for the majority of cowpea lines

ranged between 20 and 60%, 50 and 80%, and 40 and 100%, at

the three sites, respectively. Shoot dry weight at 45 days

was highly correlated with nodule dry weight, nodule

number, and ARA per plant.

As with other legumes, variable responses to starter N

and high N rates are reported for cowpea. Agboola (1978)

found 20 kg N ha-1 increased yields from 800 to 1850 and

from 1200 to 1600 kg ha-1 in soils with organic matter

contents of 5 and 10 g kg-1, respectively. No response to

starter N was obtained in soils with greater than 20 g kg-1

organic matter. In pot studies, Eaglesham et al. (1983)

found that for cowpea and soybean, applied N had a

synergistic effect on N2-fixation which depended on N

source, N rate, and cultivar.








Nitrogen fertilization (200 kg ha-1 of a 13-13-20 mix)

is recommended in Trinidad (Ferguson and Jallow, 1984) but

N fertilization of cowpea is probably not economical

(Huxley, 1980) due to the low and variable response to N

observed under most situations. Starter N rates of 12.2,

22.4, and 44.8 kg ha-1 decreased ARA, nodule number, and

nodule weight per plant. The effect was greatest on the

higher N2-fixing (C2H4) cowpea cvs. California Blackeye No.

5 and Knuckle Purple Hull than for Chinese Red (Miller et

al., 1982). Systematic drought treatments and N fertilizer

additions decreased ARA and nodule weight per plant but

yields were unaffected and averaged 2700 kg ha-1

(Zablotowicz et al., 1981). Staver (1975) found no yield

response to inoculaton or 100 kg N ha-1 in Venezuela;

yields averaged 1200 kg ha-.


Peanut (Arachis Hypoqaea)

Peanut, or groundnut, is a warm season, annual grain

legume which bears its fruit underground. Although native

to South America, it is cultivated throughout the world for

its high oil (50%) and protein (25%) content. While world

average yields are low, 880 kg ha-1, farmers in the U.S.A.

average 3000 kg ha-1 (Dart and Krantz, 1977). Since it is

relatively drought resistant and requires low inputs, it is

well-adapted to the semi-arid tropics. Peanuts are

described as runner or bunch types depending on their

branching habit. Spanish-Valencia cultivars (Arachis

hypogaea var. fastiqiata) are short-duration, bunch types








while the larger-seeded Virginia cultivars (Arachis

hypogaea var. hypogaea) are long-duration types with bunch,

runner, or more commonly, intermediate branching habits.

Nambiar et al. (1982) found that Virginia types formed more

nodules and fixed more N than did earlier-maturing Valencia

or Spanish types. Similar findings are reported by Wynne

et al. (1982). Their results showed that nodulation and

N2-fixation differences between peanut types were exhibited

primarily during early pod-fill. Leaf area duration

accounted for greater than 70% of the difference in N2-

fixation (C2H4). This is support for the finding that N2-

fixation is closely associated with photosynthetic capacity

(Hardy and Havelka, 1976). Tonn and Weaver (1981) found

that Virginia cvs. Florunner and Florigiant accumulated

more N in vegetative parts than Spanish cvs. Tamnut 74 and

Starr. Virginia types accumulated N in peanuts faster than

Spanish types and exhibited greater nodule mass and N2-

fixation (C2H4) rates.

Peanut plants are nodulated by the cowpea cross-

inoculation group of rhizobia. Although cowpea rhizobia

are generally considered present in high numbers throughout

the tropics, increased yields have been reported in certain

instances. There is evidence that inoculation response is

more likely in new land or in land under environmental

stress. Inoculation increased yields of peanut in one of

three seasons in Guyana (Chesney, 1975) on land which had

not previously been planted to peanut. The increase was

obtained in the higher-yielding (2300 kg ha-1) but not








lower-yielding (600 and 1800 kg ha-1) seasons. No yield

response to inoculation was observed by Graham and Donawa

(1982) under low-yielding conditions (500-900 kg ha-1).

Rhizobium inoculation increased peanut yield two-fold (3680

vs 1910 kg ha-1) in a sandy soil low in organic matter in

Florida (Hickey et al., 1974). Both leaf and seed N were

similarly increased in their study. In Israel, inoculation

of peanut with efficient strains of Rhizobium has increased

yields under irrigation in hot and dry seasons. Inocula-

tion of peanuts on new land enhanced nodulation and yield,

especially with adequate fertilizer additions (Albrecht,

1943). A survey of 30 sites in Sudan showed that only one

location had less than 100 cowpea rhizobia g-1 soil (Hadad

et al., 1982); high numbers were found in soils not

previously planted to peanut. No yield response to

inoculation was found for peanut in these soils. Some

response to N fertilizer was obtained under very high-


yielding conditions.














CHAPTER 3
FARM SURVEY


Soil Fertility Survey of Farms in St. Kitts

Introduction

The legume-Rhizobium symbiosis can be severely limited

by soil nutritional factors. Although soil nutrients have

little effect upon survivability of rhizobia in soils,

nutrient deficiencies can limit nodulation and subsequent

N2-fixation by affecting the growth of the legume host.

The more common nutrient constraints encountered by legumes

are associated with low pH and include low Ca, P, and Mo,

and high Al. However, any nutrient imbalance which affects

plant growth can affect N2-fixation. High soil N

negatively influences both nodulation and N2-fixation.

Therefore, soil N will play a major role in determining the

demand for N2-fixation by the legume plant.

The objective of this study was to determine soil

reaction and nutritional status of a large number of soils

from St. Kitts in order to evaluate potential soil

fertility constraints to legume production.


Materials and Methods

One hundred and eleven soil samples were collected from

farms in St. Kitts. The farms from which soil samples were








collected were not randomly selected. An attempt was made

to include farms throughout the island both at lower and

higher altitudes and to include farms on steep hillsides

and in ghuts as well as those on the less sloping ridges.

Several of the farms selected were participating in the

farming systems research project conducted by CARDI staff.

The farms were identified by the farmer's name when

possible and by the nearest sugarcane estate; estate names

were used because most farmland now or in the past belonged

to the sugarcane estates.

Since most farmers plant on raised beds, soils were

sampled from the side down 15 to 20 cm into the heart of

the bed. An attempt was made to randomly sample across the

land and to sample land under cultivation. With a few

noted exceptions, only one composite sample was analysed

per farm. The composite was a collection of at least 10

individual samples per farm. Additional information

collected included approximate altitude and predominant

slope. The altitude was classified as either low (0 to

100m), medium (100 to 150m), or high (150 to 350m) while

the slope classes were estimated as 0=flat, l=gently

sloping (0-3%), 2=moderately sloping (3-10%), 3=steep (10-

25%), 4=very steep (>25%).

The air-dried soil samples were sieved through a 2mm

screen. Soil pH was measured in a 1:2 (v/v) water suspen-

sion after at least 30 min equilibration time. Soils were

extracted with the Mehlich I (0.05 M HCl + 0.0125 M H2SO4)








extractant (1:4 w/v) as described by Rhue and Kidder

(1983). Soil extracts were analyzed for Ca, Mg, K and P by

the University of Florida Soils Testing Service (Rhue and

Kidder, 1983). Total N was determined by the Kjehldahl

procedure using salicylic acid and sodium thiosulfate to

include nitrate-N (Bremmer and Mulvaney, 1982). For the

Kjeldahl N procedure 1.00 g of air-dried and ground (5 min

on mortar and pestle) soil was used.

Additional analyses were performed on 11 selected

soils. Particle size distribution of the <2mm fraction was

determined using the standard pipette methodology (Gee and

Bauder, 1986). Clay mineralogy was qualitatively deter-

mined using x-ray diffraction on Mg-solvated clay separates

obtained by first wet sieving the soil sample through a 325

mesh (0.044mm) screen and then centrifuging in water (pH

10) for 5 min at 2000 rpm. Soil pH was measured in a 1:2

(v:v) H20 suspension. Calcium, Mg, Na, and K were

determined in a 1:10 (w:v) ammonium acetate (pH 7) extract

(Thomas, 1982). Phosphorus was determined in Mehlich I as

described earlier. Aluminum was extracted in 1 M KC1 and

titrated with NaOH to a phenolpthalein endpoint (Thomas,

1982). Total Kjehldahl N (TKN) was determined as

previously described and organic C was determined by the

Walkley-Black method (Nelson and Sommers, 1982).


Results and Discussion

A summary of results from the soil fertility survey is

presented in Tables 3-1 and 3-2; a complete listing of the








Table 3-1.


Classification of 111 soils from farms surveyed
in St. Kitts according to Mehlich I extractable
K and P.


Distribution of soils in classes


Cumulative

Soil Sufficiency Range No. %
nutrient class$ No. %


- mg kg-1 -

K vlow (0-18) 0 0.0 0 0.0
low (19-36) 31-36 2 1.8 2 1.8
med (37-62) 52-56 4 3.6 6 5.4
high (63-123) 63-121 40 36.0 46 41.4
vhigh (124- ) 124-482 65 58.6 111 100.0


P vlow (0-8) 4-8 22 19.8 22 19.8
low (9-16) 9-16 33 29.7 55 49.5
med (17-29) 18-29 27 24.3 82 73.8
high (30-59) 30-56 18 16.2 100 90.0
high (60- ) 61-293 11 10.0 111 100.0


$ vlow = less than 50% of crop yield potential is
expected without addition of the nutrient;
low = 50-75%; med = 75-100%; high = sufficient;
high = greater than adequate, could cause nutrient
imbalances if nutrient is applied.








Table 3-2. Classification of 111 soils from farms surveyed
in St. Kitts according to soil N and soil pH.


Distribution of soils in classes


Cumulative

Soil Class No. %
factor range No. %


pH 5.0-5.5 4 3.6 4 3.6
5.6-6.0 31 27.9 35 31.5
6.1-6.5 44 39.6 79 71.1
6.5-7.0 18 16.2 97 87.3
7.1-7.5 10 9.0 107 96.3
7.6-8.0 2 1.8 109 98.2
8.1-8.5 2 1.8 111 100.0


N 0.50-0.75 12 10.8 12 10.8
(g kg-1) 0.76-1.00 34 30.6 46 41.4
1.01-1.50 56 50.5 102 91.9
1.50-2.00 4 3.6 106 95.5
2.01-5.00 5 4.5 111 100.0


1:2 soil:water (v:v)
Total N including nitrates








soils and individual results from chemical analyses are

provided in Appendix A. The soils were grouped into

sufficiency classes according to Mehlich I extractable P

and K, and into arbitrary classes according to pH and TKN.

Of the three major nutrients, P presented the greatest

potential for a nutrient deficiency problem. Half of the

soils sampled tested either low (9-17 mg kg-1) or very low

(<9 mg kg-1) in P according to guidelines established by

Rhue and Kidder (1983) of the University of Florida's

Extension Soil Testing Laboratory for agronomic crops;

critical levels established for vegetable crops are higher.

Soil test P was related to altitude so that 5.3, 48.3

and 76.5% of soils tested low or very low in low, middle,

and high altitudes, respectively. This decrease in soil

test P with increasing altitude is a consequence of the

more highly weathered and finer-textured soils found at

these higher elevations. Mean annual rainfall based on 30-

year averages is 132, 148, and 151 cm for low, middle and

high zones, respectively. Higher rainfall coupled with

cooler temperatures (less evaporation) have resulted in

greater weathering of soils at the higher elevations. Only

at higher elevations can one find reddish-brown silt loams

and clay loams. As discussed in a later section, the clay

fraction of these finer-textured soils is largely amor-

phous. Amorphous minerals associated with soil of volcanic

origin are known to be low in available P due to high P

retention/fixation properties (Sanchez and Uehara, 1980;








Yuan, 1974). Since few farmers use fertilizers, P defi-

ciency may limit potential benefits derived from successful

inoculation and subsequent N2-fixation. Clearly, future

agronomic studies with P are warranted.

Soil test K levels were high or very high in 95% of the

soils tested. The high soil K levels observed throughout

the island and on farms where little or no fertilizer is

used indicate a K-rich parent material. These results

indicate that K would not be a nutritional constraint to

legume nodulation and N2-fixation.

Soil pH was found to be generally favorable throughout

the island. Of the soils tested, 84% had a pH in the

optimal range of 5.5 to 7.0. Only 4% of the soils had a pH

less than 5.5, the approximate value below which Al+3

activity becomes detrimental. Soil Ca ranged from 780 to

2280 mg kg-1 and was correlated with pH (r=0.52). These

relatively high soil Ca levels coupled with the favorable

soil pH's would indicate that Ca poses no threat as a

potential limiting nutrient.

Due to greater rainfall and weathering conditions, soil

Ca and pH decline at the higher elevations. Thus, the

average pH was 6.8, 6.3, and 6.1 and the average soil Ca

1530, 1200, and 1160 mg kg-1 for soils from low, middle,

and high altitude zones, respectively. Soil test Mg was

sufficient and averaged 300, 280, and 260 mg kg-1 for soils

from low, middle, and high zones, respectively.






44

Certain soils around the island had relatively high pHs

(>7). Soils with a pH greater than 7.0 were observed in

26% of soils sampled from the low altitude zone, 7% from

the middle zone, and 5% from the high zone. Two soils were

visibly calcareous and exhibited soil pH's of 8.3 and 8.4.

For soils having a pH greater than 7, micronutrient defi-

ciency problems should be considered. The only soil

micronutrient analyses made were for the soil at the CARDI

Research Station which had a pH of 7.2. For this soil

Mehlich II (0.2 M NH4Cl + 0.2 M HOAc + 0.015 M NH4F + 0.012

M HC1, pH 2.5) extractable Zn, Cu, Mn, and Fe were 10, 4,

67, and 2 mg kg-1, respectively. Critical levels for Zn,

Cu, and Mn in Mehlich II extract are 4.8. 0.8, and 1.2 mg

kg-1 (Mehlich, 1978). According to these sufficiency

levels, the soil at the CARDI Research Station tested high

for Zn, Cu, and Mn.

Total Kjeldahl N is a measure of the N reserve of a

particular soil. For soils which have not recently

received considerable N fertilizer, approximately 95% of

this N is a component of the organic matter complex and as

such is only available upon mineralization. Assuming an

annual mineralization rate of 2% year-1 (Barber, 1984) and

2 x 106 kg soil ha-1, the amount of inorganic N released

through mineralization would be 40, 80, 200 and 400 kg N

ha-1 year-1 for soils with 1, 2, 5, and 10 g TKN kg-1.

Since a growing season is seldom longer than 2 to 3 months

and the period of maximum N uptake even less, positive








response to N fertilizer application is expected for most

crops on soils with TKN's less than 2 g kg-1 soil.

Total Kjeldahl N for the 111 soils tested ranged from a

low of 0.53 g kg-1 to a high of 4.62 g kg-1. Despite the

wide range, 79.3% of all soils had TKN's between 0.76 and

1.50 g kg-1. Only five of 111 soils had TKN's greater than

2.0 g kg-1 and these were all from the high altitude zone.

Mean TKN was 1.37, 1.02, and 1.05 g kg-1 for soils from

high, middle and low zones, respectively. The low soil N

values indicate that the majority of soils tested would not

provide adequate N for acceptable production of agronomic

crops. Furthermore, a leguminous crop would be expected to

depend upon symbiotic N2-fixation for much of its N

requirement.

Additional information obtained for eleven selected

soils is given in Tables 3-3 and 3-4. These 11 soils,

which were collected from farms that have participated in

various agricultural experiments, were representative of

the soils around the island. Soil texture ranged from the

more common sandy loam to silty loam. All soils had 10% or

less by weight of clay. The sand size distribution was

approximately normal for all 11 soils with medium sand

being the most abundant.

The silty loam soil was found at a location called

Phillips Level which is a high elevation (1000 m) pass

through the two main volcanic ranges. The area has been

used by the government for citrus and coffee production.













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Aluminum extracted in 1 M KC1 was noticeably greater for

this soil than for the other soils tested but the level was

still low. Using the sum of the basic cations extracted by

ammonium acetate plus the KCl-extractable Al as the

exchange capacity of the soil, percent Al saturation in the

Carty soil was 0.28/(8.27+0.28) or only 3.3%. Depending on

the tolerance of the crop, Al toxicity does not generally

become a problem until percent Al saturation reaches 20 to

30%. High Al saturation is associated with soils which

have pH's below 5. Since this farm is representative of

the most highly weathered soils on the island, low pH and

associated problems of low Ca and high Al saturation are

not constraints to legume production in St. Kitts.

The soil at CARDI had a pH of 7.2. The high pH was due

in part to the relatively high extractable Na conc. The

source of the Na was most likely the irrigation water.

Very low levels of Na were observed for the other soils.

The Stuffy soil at West Farm estate also had a high pH.

The high pH was not due to Na but rather appears to be

related to a Mg-rich component. The Ca:Mg ratio for this

soil was only 1.2 versus 2.9 for the other 10 soils.

Mehlich I extractable P was high for CARDI, Huggins,

Herbert, and Caesar soils. Fertilizer is routinely used on

each of these farms thereby resulting in the unusually high

P levels. Both Herbert and Caesar farms have recently been

using chicken manure obtained from a local layer operation.








As discussed in the general soil fertility survey,

total N for most of the soils was low (less than 1.5 g

kg-1) increasing somewhat at higher elevations. The soils

at higher elevations (Carty, Langley, Caesar) were also

associated with higher organic C contents and lower pH's.

High soil N and organic matter content may reduce the

dependence of legumes on deriving N from symbiotic N2-

fixation at the Carty farm at Phillips Level. On the other

hand, some research has demonstrated that the organic

matter fraction associated with weathered volcanic soils

may be more stable and exhibit reduced mineralization rates

due to strong completing of the organic matter to the

amorphous minerals (Yuan, 1984).

X-ray diffraction was used to identify crystalline

minerals in the minus 325 mesh (.045 mm) fraction of the 11

soils. Two x-ray diffractograms are presented in Figures

3-1 and 3-2. Although a small peak at a d-spacing of 4.4 A

would indicate a small amount of smectite in the Carty silt

loam soil, the absence of crystalline minerals was clearly

evident in all 11 soils. Broad, diffuse peaks with d-

spacings from 3.5 to 3.0 were observed for all soils and

may indicate an amorphous or weakly crystalline clay.


Summary and Conclusions

A soil fertility survey of 111 soils from farms in St.

Kitts was conducted to assess potential nutrient con-

straints to legume production. Analyses performed on these





























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soils included pH, TKN, and Mehlich I extractable Ca, Mg,

K, and P.

Extractable soil P was rated deficient or marginally

deficient in 74% of the soils tested. That soil P was high

in a few soils which received P fertilizer is evidence that

P deficiency is related to a lack of P fertilization rather

than to a severe P fixation problem. Results indicated

that of all the nutritional problems to consider, P

deficiency poses the greatest potential to limit legume

production in St. Kitts. It follows that P fertilization

should be incorporated in future production and research

programs.

Soil test Ca, Mg and K concs were, with few excep-

tions, more than adequate for legume production. High soil

test K values were found throughout the island and indicate

that the volcanic parent material is rich in potassium-

bearing minerals.

Soil reaction was generally favorable with 84% of the

soils having pH's between 5.5 and 7.0. Due to greater

rainfall and therefore more intense weathering conditions,

average soil pH was lower at the higher elevations (6.8)

than at the lower (6.1) elevations. No soil was found to

have a pH less than 5. It follows that few, if any, soil

acidity problems should be encountered in the island.

The majority of the soils were low in N. Higher TKN

contents were observed for soils at higher altitudes. The

higher N contents were attributed to the presence of more








highly weathered soils containing amorphous clay minerals

which complex the organic matter and decrease minerali-

zation rates. The latter statement is supported by x-ray

diffraction analyses of several representative soils which

showed little if any crystallinity in the silt and clay

size fractions. Regardless, the deep, well-drained nature

of the majority of the soils of St. Kitts would indicate

that N leaching would be sufficient to prevent accumulation

of high levels of inorganic N. It was concluded that

response to N fertilizers or N2-fixation would be expected

for legumes grown in most soils of St. Kitts. This is not

surprising since few agricultural soils, muck soils being a

notable exception, can sustain crop production without some

form of N input.


Rhizobium Survey of Farms in St. Kitts

Introduction

Populations of Rhizobium species vary from one location

to another. Factors affecting their number and

distribution in soils include the legume-cropping history

of the soil, soil organic matter content, soil temperature,

soil moisture, and soil texture (clay content and clay

mineralogy). The distribution of Rhizobium species has not

been investigated in St. Kitts.

Several methods of investigating rhizobia distribution

in soils of St. Kitts were considered. A popular method

which quantifies rhizobia is the most probable number (MPN)

methodolgy whereby serial dilutions of each soil are used








to inoculate legume seedlings grown in plastic growth

pouches, test tubes, or any containers which can be

rendered rhizobia-free. If many soils and several legume

species are being investigated, the number of these

containers soon becomes considerable. Another method is to

seed legume species directly into the soil and observe

nodulation after several weeks of plant growth. As with

MPN methods, inoculated controls are included to assure

that conditions are conducive to infection and nodule

formation. Although these grow-out tests could be per-

formed in situ, soils are usually removed for testing under

controlled conditions to insure adequate legume growth. A

standard inoculation study utilizing uninoculated and N-

fertilized controls can also be useful in determining the

presence of rhizobia in soils. As with the MPN method, the

standard inoculation trial quickly becomes considerable in

size when more than a few soils are investigated. There-

fore, this method is utilized when effectiveness is a

primary concern.

The purpose of this study was to determine if cowpea,

soybean, and bean rhizobia were present in selected soils

from St. Kitts and to assess the effectiveness of

indigenous bean rhizobia in relation to a known effective

strain and N application. To this end soils were gathered

from farms in varying locations in St. Kitts and evaluated

in pot culture. Qualitative grow-out tests were run for









soybean and cowpea rhizobia while standard inoculation

trials were conducted for bean rhizobia.


Methodology

Soils from 35 farms were included in this study. Soil

composites were collected from each site and transported to

CARDI in plastic garbage bags. The soils were sieved

through a 3 mm screen to remove stones and debris; soils

were not air-dried. The screen was flamed with alcohol in

between soil sievings to prevent cross-contamination.

A subsample of each soil was sieved through a 2mm

screen and transported to the University of Florida for pH

(1:2 v:v H20) and TKN determinations.

Cowpea cv. California Blackeye No. 5 and soybean cv.

Jupiter seed were sown into moistened soils placed in

duplicate 10 cm tall (400 g capacity) plastic cups. Inocu-

lated controls cowpeaa strain TAL369 and soybean strain

NIT61A142) were included for each soil-legume combination.

Rhizobium strains were cultured separately in 3 ml YMB

(Appendix B) until a turbid suspension was obtained. Five

drops of inoculum were placed at the bottom of each seed

hole just before planting. After emergence, plants were

thinned to two per cup and placed outside under 55%

shadecloth. Nodulation was noted after 18 days of plant

growth.

Replicated inoculation trials were conducted to

evaluate soils for the presence and effectiveness of bean

rhizobia. The three treatments applied to each soil in








three replications were

1) uninoculated control,

2) inoculated with bean strain Nitl27k44, and

3) N at 50 mg/kg as urea.

Soils were tested in three groups of 12. Soil from the

CARDI Research Station was sterilized in the pressure

cooker for 3 hours to be included as a control medium to

monitor contamination problems. Plants grown in this

autoclaved soil died rapidly after emerging. No further

attempt was made to include such a control. Solutions to

provide 1.0 g OSP (20% P205), 0.2 g KC1 (60% K20), and 0.04

g micronutrient mix were added to 2000 g of soil in plastic

bags. A urea solution was added along with the other

fertilizers for the N treatment. Small increments of water

were then added to bring the soil to a moist soil

condition. A black polyethylene bag was then filled with

the 2000 g of fertilized soil. Seeda of bean cv. Miss Kelly

were surface-sterilized in 80% Chlorox (4% calcium hypo-

chlorite) for 5 min and planted immediately after rinsing

with water several times. Bean strain Nitl27k44 cultured

in YMB was added to the planting hole at 5 drops per hole.

In order to provide a moisture-conserving mulch and to

prevent splashing of soil from one pot to another, the

surface of each pot was covered with a 1 cm layer of 3-5 mm

gravel autoclaved for 2 hours in the pressure cooker.

Plants were thinned to 3 per pot by cutting the base of the

stems. After 4 to 5 weeks of growth outside under 55%






57

shadescreen, whole plants were dried, weighed, and analyzed

for N. Due to the large number of nodules on bean roots,

nodules were not counted and weighed. Instead, observa-

tions relative to abundance, color, and size were made. A

randomized complete block design with three replications

was used to evaluate treatment effects for each individual

soil.


Results and Discussion

Identification and selected properties of the 35 soils

used in this study are presented in Table 3-5. Soil pH

ranged from 5.5 to 8.3 and soil TKN ranged from 0.6 to 3.9

g kg-1. Variations in plant growth observed between the

different soils were due in part to soil textural differ-

ences and associated water relations; some reduction in

growth was noted in finer-textured soils due to poorer

drainage conditions.

Plant dry weight yield and total N accumulation of

beans are presented in Table 3-6. The growth and N accumu-

lation response from Rhizobium inoculation and N applica-

tion varied widely among the different soils (Figure 3-3).

Rhizobium inoculation increased plant dry weight in 11 of

the 35 soils tested. However, no increase in plant dwt

from N application was observed in 5 of the 24 soils in

which no response to inoculation was observed. Therefore,

Rhizobium inoculation increased yields in 11 of 30 (37%)

soils which were responsive to N application. Nitrogen









Table 3-5.


Identification and selected properties of soils
collected from farms in St. Kitts for evaluation
in pot culture studies.


No. Location Altitudel Textural pHl# No
class


Liburd, West Farm
West Farm
Depaussant, West Farm
James, Olivees
Liburd, Fountain
Williams, Fountain
Alfred, Olivees
Armstrong, Camps
Huggins, Fountain
nn, Trinity
Smithen, Cunningham
nn, Bayford
nn, Wingfield
Bromne, Camps
Arthur, Gauchet
DOA, La Guerite
nn, Trinity
Powell, Hermitage
nn, Upper Spooners
Woodley, Lamberts
nn, Canada
nn, Lynches
nn, Greenhill
Caesar, Greenhill
nn, Fahies
nn, Pogson
nn, Sandy Point
Lawrence, Lynches
nn, Halfway Tree
nn, Halfway Tree
nn, Belle Tete
nn, Phillips
Morris, Phillips Level
Thomas, Phillips
CARDI, Basseterre


low
high
middle (ghut)
high
high
high (ghut)
middle (ghut)
high
middle
middle
high
high
high
low
high (ghut)
low
low
middle (ghut)
high
middle
low
middle (ghut)
high
high
middle
middle (ghut)
low
middle
middle (ghut)
middle
low
high
high
high
low


$ Low (0-75m); middle (75-150m); high (>150m)
$$ 1:2 (v/v) water suspension
p Total Kjeldahl N including nitrates
nd = Not determined
nn = No name provided


ls
1
Is
ls
sl
ls
Is
s
1
Is
Is
sil
sl
sl
Is
sl
ls
Is
sl
Is
Is
sl
sl
sl
Is
Is
sl
sl
Is
Is
sl
sl
sil
sl
1









Table 3-6.


Growth and N content of pot-grown beans in
selected soils of St. Kitts as affected by
Rhizobium inoculation and N application.


Plant dry weight N content


Soil -Inoc +Inoc N LSD -Inoc +Inoc N LSD


- g/3 plants -


5.7
6.2
5.6
2.5
6.8
4.6
3.6
5.7
3.5
4.5
3.5
3.8
4.3
3.2
2.9
4.2
4.3
5.4
2.5
4.5
5.5
4.5
6.3
8.5
4.1
5.5
5.4
5.8
5.4
6.0
4.3
10.3
7.2
5.4
5.0


6.9*
9.0*
5.4
2.5
6.3
5.3*
3.9
7.3*
5.7*
5.2
2.9
3.3
4.2
3.0
4.2*
4.4
4.7
5.7
2.4
4.8
5.4
5.6*
5.5
9.9*
6.2*
6.2
5.8
5.7
6.4
7.9*
5.1*
11.0
7.9
5.6
4.8


8.3*
9.9*
7.0*
6.4*
7.1
6.8*
7.0*
8.4*
6.4*
7.1*
5.1*
3.8
5.4*
4.4*
3.9*
4.7
6.9*
6.0
5.9*
6.1*
5.7
7.3*
8.2*
9.8*
9.1*
8.3*
8.5*
8.3*
7.9*
9.6*
7.6*
11.7*
9.1*
7.5*
6.5*


- mg/3 plants -


0.21
0.71
0.72
0.71
0.78
0.41
0.36
0.67
1.03
0.80
0.78
1.14
0.40
0.34
0.59
0.88
0.82
0.77
0.31
0.44
0.47
0.26
1.02
0.74
0.51
0.83
0.77
0.61
1.03
0.98
0.67
0.75
1.10
0.37
0.57


142*
189*
153
61
158
139*
95*
157*
146*
119
64
93
95
102
129*
137
139
120
54
128
141
162*
158
214*
159*
143
147
160
187*
104*
141*
279
190
139
132


202*
220*
197*
155*
191
167*
128*
171*
156*
133
103
78
113
89*
115*
129
199*
154*
111*
138
162
169*
236*
232*
201*
184*
195*
143*
191*
157*
170*
295
182
170*
160*


* Significant increase over the
according to LSDO.05-


uninoculated control







60



-I-





j .4J
mul 0
F- 4J
mm>








-q-
I U -JO
OD I Ul





s mN C o


N *3 1.






mU o o

mmN 0





14.1 i.4^ 4 )
. '









H ,-4-1





'l -l a4
0 ,--


CUN


(0our- JaAO asUG=Urr %) asMOdsaI cnaI2 UIDIM MaC LMVId


O O u 0
Ns ID In rn






61

content of the bean plants was increased by inoculation in

13 of 35 soils; plants in two soils (nos. 7 and 29) had

increased N contents although plant dry weight was not

increased.

Nodulation of uninoculated bean plants was great

(greater than 100 nodules per plant) in all soils with a

few exceptions. In soil nos. 19, 25, 29, 30, and 31,

control plants had very few (<20) nodules; only 2 nodules

per plant were found in soil no. 19. Four of these five

soils are from the north end of the island, no. 19 being

the exception. Nodules in soil nos. 25 and 29 were small

and white, in no. 30 they were large and green or small

and white, and in 31 they were pink. Small white nodules

can be either immature, developing, or be the result of

infection by ineffective strains; a relatively high

proportion of these small, white nodules indicates the

latter. Green nodule interiors are evidence of a senescent

nodule. A high proportion of these nodules can also be an

indicator of ineffective strains but more often indicates a

stressed plant which can no longer support these nodules.

The great majority of nodules formed in the various soils

tested were pink-red nodules which result from effective

nodulation. Of the five soils with poor nodulation, only

in soil no. 19 was an increase in plant dry weight yield

from inoculation not observed. It appears some other

unknown factor was limiting to N2-fixation in soil no. 19

since the plant was responsive to N addition. Uninoculated








plants in soil nos. 8, 9, and 15 had relatively high

percentages (25 to 50%) of green nodules; an increase in

plant dwt and N content from inoculation were observed in

each of these three soils.

There did not appear to be any relationship between

response to inoculation and soil pH or soil N. Of the 11

soils which responded to inoculation, pH ranged from 5.8 to

7.6 while soil N ranged from 0.7 to 1.5 g kg-1. Total N

averaged 1.11, 1.24 and 1.08 g kg-1 for soils which were

responsive to inoculation, unresponsive to inoculation, and

unresponsive to N or inoculaton, respectively. Soil pH

averaged 6.4 for each response group. Altitude, which is

related to rainfall and temperature, may help predict areas

where nodulation may be more important. Beans in 2 of 8

(25%), 5 of 12 (42%), and 4 of 15 (28%) soils responded to

Rhizobium inoculation in low, middle, and high zones,

respectively.

Cowpea nodulation was observed in all but soil no. 12.

This soil was a poorly drained silt loam which tended to

become waterlogged in the plastic cups. Since only a few

nodules formed in this soil despite inoculation, the

possibility exists that under better drainage conditions

nodulation from indigenous rhizobia may occur. No soybean

nodules were observed on uninoculated Jupiter plants in any

soil. Inoculated controls were well-nodulated indicating

that indigenous soybean rhizobia were absent or that they

were present in very low numbers.









Summary and Conclusions

Soils were collected from thirty-five farms throughout

the island to investigate the distribution of indigenous

soybean, cowpea and bean rhizobia. For soybean and cowpea,

this encompassed growing out respective legumes in the

potted soils and qualitatively assessing nodulation after

several weeks of growth. In order to evaluate effective-

ness of indigenous bean rhizobia, a replicated inoculation

trial was conducted for each soil.

Nodules were formed on cowpeas grown in 34 of 35 soils.

No nodules were observed in a silt loam soil that was

poorly drained. No uninoculated soybean plants were

nodulated when grown in the 35 soils. Since inoculated

controls were well nodulated in each soil, populations of

soybean rhizobia were very small or non-existent. These

results were not unexpected as nodulation of native legumes

is prevalent throughout the island. The absence of soybean

nodulation was also expected but for another reason.

Jupiter soybeans have specific requirements for B.

japonicum. Since soybean cultivation has not practiced in

St. Kitts, this specie would not be expected to be

established in soils in the island.

The growth of the bean cv. Miss Kelly was quite

variable in the different soils and under different

inoculation treatments. An increase in dry weight yield

from inoculation relative to uninoculated plants was

observed for 37% of the soils which also responded to N








application. No increase in growth from inoculation or N

fertilization was observed for 5 of 35 or 14% of the soils

tested. Nitrogen content was increased in two soils in

which no dry weight increase was observed. Plant dry

weight and N content were positively correlated (r=0.80).

Plant dry weight yield or visual ratings have proven to be

invaluable parameters for evaluating inoculation success in

large screening programs (Halliday, 1983).

No relationships were observed between several soil

properties and response to inoculation. The inability to

predict whether a particular soil-cultivar situation will

respond to inoculation has always been a major problem for

beans and is one of the many reasons why N fertilization is

a common practice for bean production. Also, response to

inoculation in pots in no way guarantees a similar response

in the field. In fact, a bean inoculation field trial

conducted at Huggins' farm indicated no benefit from

Rhizobium inoculation despite a positive response for the

same soil in this pot study.
















CHAPTER 4
FILTER-PRESS MUD AS AN ALTERNATIVE INOCULANT CARRIER


Introduction

In the event that local production of legume inoculants

is warranted, production technology utilizing locally

available materials would be desirable. Since peat is not

available in St. Kitts, the selection of an alternative

carrier material is important. Potential alternative

inoculant carriers identified in St. Kitts included char-

coal, bagasse, and filter mud; coconut coir dust was

available on Nevis. Each of these materials has been found

to support the growth and survival of rhizobia when

properly processed and inoculated with broth cultures of

Rhizobium (Faizah et al., 1980; Philpotts, 1976; Ryder and

Grant, 1983; Sparrow and Ham, 1983).

Filter mud, as it is hauled away from the sugarcane

processing plant to the dump, contains too much sugar to be

directly used as a carrier material. Plants will

plasmolize if planted directly into fresh filter mud

amendments. Many local gardeners have filter mud delivered

to their home gardens where it is stockpiled and allowed to

compost. After some time, usually several months to a

year, the filter mud is incorporated into the garden soil

as an organic soil amendment and as a fertilizer source.

65








Filter mud has been shown to be an excellent medium for

Rhizobium growth and survival. Before filter mud could be

recommended as a potential inoculant carrier material for

St. Kitts, an assessment of the suitability of locally

available filter mud was needed. Therefore, the objective

of this study was to monitor the growth and survival of two

commercial Rhizobium strains, a slow-growing soybean strain

and a fast-growing bean strain, in filter mud carrier as

compared to that observed in standard commercial peat

carrier.


Materials and Methods

The filter mud chosen for inoculant carrier studies

was obtained from an approximately 1-year old pile which

had been exposed to the elements during that time. The

filter mud was air-dried and ground in a Wiley-type mill to

pass a 1-mm screen. To produce powdered seed inoculant,

the ground filter mud was further screened through a no.

100 mesh (0.15 mm) sieve. Commercial class 2 peat was

obtained from Dr. Stewart Smith of the NITRAGIN Company,

Milwaukee, Wisconsin.

Characterization of the two materials was conducted at

the University of Florida. The pH of both materials was

determined in a 1:2 (v:v) water suspension. Organic matter

content was determined as the percent loss on ignition

(5000C). Mehlich I (0.05 M HC1 + 0.0125 M H2SO4)

extractable K, Ca, and Mg were determined as previously

described for soils. Stacked sieve analyses were made on








both the original ground (<1 mm) and minus no. 100 sieve

fractions of filter mud. Moisture content at field

moisture capacity (33 kPa or 0.33 bar) was determined using

the porous plate methodology (Cassel and Nielsen, 1986).

An incubation study was conducted to evaluate the

growth and survival of two commercial NITRAGIN CO. strains

of Rhizobium, Nitl27k44 (fast-growing bean strain) and

Nit61A142 (slow-growing soybean strain), in peat and filter

mud carriers over a 2-month period. The two strains were

added individually to autoclave-sterilized filter mud and

peat materials to formulate single-strain inoculants. The

peat was neutralized by the addition of 7 g of precipitated

CaC03 per 100 g of peat prior to use.

Inoculants were produced by inoculating the carriers

with yeast mannitol broth (YMB) (Appendix B) cultures of

the individual strains so that the final moisture content

was approximately 40% on a wet weight basis (wwb). This

moisture content was found to give the best physical

characteristics of both peat and filter mud, and was

similar to the field moisture capacity at which rhizobial

growth and survival has been found to be optimal; addi-

tional moisture resulted in undesirable clumping of the

material. The high absorption of filter mud, despite

having less than half the organic matter content of peat,

is due mostly to the fine bagasse fibers which alone have

been reported to absorb over ten times their own weight

(Ryder and Grant, 1983).








After some trial and error, the 40% moisture in the

final filter mud product was best accomplished as follows.

To 150 g of the air-dried filter mud (10% wwb) and neutral-

ized peat (12% wwb), 20 mL of water were added and mixed in

before autoclaving. Autoclavable 12.5 x 17.5 cm

polypropylene bags (Bel-Art Products, New Jersey) were used

for packaging. The thickness of the polypropylene film was

0.05 mm. After autoclaving the materials in partially

sealed (autoclave tape) polypropylene bags for 2 hours at

100-110 kPa, the bags were allowed to cool overnight inside

the autoclave. The following morning the bags were sealed

using adhesive tape. After autoclaving, moisture content

was 19 and 23% wwb for filter mud and peat, respectively.

The two strains were cultured separately in -2 L glass

fermentors containing 500 mL of YMB. At the base of each

fermentor was an aspiration port from which samples were

periodically drawn for microscopic examination using the

Gram-stain technique (Vincent, 1970). The fermentors were

continuously aerated via an aquarium pump system

(Somasegaran et al., 1982). After 8 days, the broth

cultures were very turbid and ready for use.

A total of 30 mL of YMB were added to each bag of pre-

sterilized carrier via a 50 mL autoclave-sterilized syringe

fitted with a 18 gauge hypodermic needle. The puncture

hole was covered with a small label. Mixing of the broth

and carrier was done by massaging the bags by hand. Twelve

bags of each strain-carrier combination were produced so







69

that duplicate bags could be sampled after 0, 2, 4, and 8

weeks of incubation. Inoculant bags were stored at room

temperature (26-290C). The number of rhizobia in the broth

inocula was determined using the Miles-Misra drop plate

technique (Hoben and Somasegaran, 1982; Somasegaran et al.,

1982). This method utilizes pipettes calibrated to deliver

a drop of known volume (0.027 mL). Ten drops per dilution

were delivered onto yeast manitol agar containing Congo red

(Appendix B). The average number of colonies was calcu-

lated from dilution plates where individual colonies could

be distinguished.

The number of rhizobia was determined in duplicate bags

of each carrier-strain combination after 0, 2, 4, and 8

weeks of incubation. Viable numbers of rhizobia were

quantified as follows. From each bag, 10 g of inoculant

were aseptically transferred to 90 mL of sterile water in a

125 mL Erlenmeyer flask. The stoppered flasks were shaken

for ten minutes and ten-fold diution series made. The

drop-plate method as described above was used to enumerate

rhizobia. The average count of the duplicate bags is

reported. Percent moisture, which was determined on a 10 g

sample dried at 1000C, and pH, which was determined as

previously described, were also measured at each date.









Results and Discussion

Characteristics of the two inoculant carrier materials

are presented in Table 4-1. While peat had a natural pH of

4.5 and required neutralization with CaCO3, filter mud had

a pH of 7.2 and, therefore, did not require any pH-modi-

fying amendments. This is an important benefit as finely

divided lime is not locally available. Philpotts (1976)

and Khonje (1983) both reported certain filter mud batches

to have pH values equal to 8.3 due to the addition of lime

in the sugarcane clarification process. However, growth


Table 4-1. Selected characteristics of the peat
mud carrier materials.


and filter


Character NITRAGIN peat Filter mud


pH (H20) 4.5 7.2
Organic matter (g kg-1) 873 388
Percent moisture (wwb) @ 33 kPa 43 39
Mehlich I-extractable
(mg kg-1)I: P 3.3 9
K 11.2 182
Ca 52.1 3200
Mg 11.4 376
Mn nd 13
Cu nd 0.8
Fe 21.0 1.2
Zn nd 2.0

Sieve analysis: % On 100 mesh 5 3
% On 200 mesh 13 74
% Through 200 mesh 82 23


$ Ash composition (g kg-1) for
nd = Not determined


peat (from Burton, 1979)









of rhizobia in these high pH filter mud-based inoculants

was not adversely affected.

The peat carrier had a higher organic matter content

than the filter mud. The higher mineral content of the

filter mud compared with peat (61 vs 13%) is due to the

fine soil particles that are associated with the hand-

harvested cane.

Percent moisture retained at a tension of 33 kPa,

commonly referred to as field moisture capacity, describes

the capacity of the material to hold water at the optimal

tension for rhizobial growth. In this respect, filter mud

compared favorably with peat. The 39% moisture at 33 kPa

observed for filter mud was similar to 38% reported by

Khonje (1983). The greater the amount of water held by a

carrier material, the greater the amount of broth inoculum

that can be initially added to the carrier.

Results depicted in Figure 4-1 indicate that the

filter mud was favorable for rhizobial growth and survival.

Although there was some reduction in viability relative to

the peat standard during the second month, the counts were

still high (greater than 8.5 logl0 rhizobia g-1) after 8

weeks. The normal practice of refrigerating inoculants

after 2 weeks of initial incubation should improve long-

term viability, although this can depend upon the strain

and moisture content (Thompson, 1980).

Similar counts were expected at day 0 since identical

volumes of broth were added to each carrier. The higher







72




Filter mud

10.0 / 9.84
9.55 9.32
1.. 9.27
o 9.0 -,-"'* 9.31
8.31 8.78
8.0 8.53

Peat



6.0
0 2 4 8
WEEKS




10.0 Filter mud

9.11
8.90
9.0 -859 8.78
S8.58

8.0 8.31 8 8.38
831 8.38 Nit61A142 (slow-grower)

S7.0 peat


6.0
0 2 4 8
WEEKS




Figure 4-1. Viability of two Rhizobium strains in
presterilized inoculants made from peat and
filter mud and kept at 26-290C during a
8-week period.









counts found in filter mud relative to the peat at day 0

may have been due to greater absorption of the broth by the

peat particles. Ideally, all rhizobia are dispersed during

the shaking procedure. Dispersion may have been more

complete in the filter mud.

The broth inoculum contained 9.63 log10 rhizobia mL-1

and 9.13 logl0 rhizobia mL-1 for strains Nitl27k44 and

Nit61A142, respectively. The lower count for Nit61A142

inoculum accounted for the lower inoculant counts observed

for this strain at day 0.

Moisture and pH during the incubation period are

presented in Table 4-2. Loss of moisture during the 8-week




Table 4-2. Moisture and pH of inoculants made from peat and
filter mud during eight weeks of incubation at
room temperature (26-290C).


Percent moisture pH


Weeks Weeks
Inoculant/
strain
0 2 4 8 0 2 4 8


Filter mud - - % - -

Nitl27k44' 39 40 35 33 7.2 7.4 7.2 7.3
Nit61A142 37 39 36 31 7.3 7.3 7.2 7.3

Peat

Nitl27k44 38 39 37 34 7.3 7.3 7.2 7.3
Nit61A142 40 40 39 36 7.3 7.3 7.3 7.3


4 Nitl27k44 is a fast-growing bean
slow-growing soybean strain.


strain; Nit61A142 is a









period was experienced for both materials. Moisture

declined slightly more for filter mud but was still 32%

(wwb) after 12 weeks. The water pH of both materials

remained unchanged at 7.2 to 7.3 throughout the incubation

period. Philpotts (1976) found that autoclaving the filter

mud reduced the pH of the material from 8.1 to 6.2.

Autoclaving had no effect on the pH of the filter mud from

St. Kitts.


Summary and Conclusions

The potential for using locally available filter mud as

a carrier for Rhizobium inoculants was investigated. The

growth and survival of two Rhizobium strains in autoclaved

filter mud compared favorably with the standard peat

carrier. After 8 weeks, counts of both the fast-growing

bean strain and the slow-growing soybean strain in the

filter mud were greater than 8.5 logl0 rhizobia g-1 inocu-

lant. Although these counts were less than those observed

for peat, the differences were on an order of magnitude

less than five.

The above experimentation was not meant to be a

comprehensive evaluation of filter mud as a carrier

material; other studies have shown it to be an acceptable

material. Instead, the study was undertaken to determine

if filter mud from St. Kitts had any peculiar properties

that might prevent its use in a local production program.

With some initial trial and error, a procedure was found

which allowed for presterilized local filter mud to support







75

high populations of both a slow-growing and a fast-growing

strain. If and when an inoculant program is initiated,

these results indicate that filter mud is a viable alterna-

tive to peat. This is not to say that other potential

carriers do not exist in St. Kitts. Sugarcane bagasse has

potential but requires neutralization (pH of 5.1 in St.

Kitts). Charcoal is extremely messy to work with and is

relatively expensive. Coconut coir dust is available only

on the island of Nevis. In the event filter mud becomes

unacceptable for local production conditions, these

materials should be investigated. However, the free and

abundant supply, coupled with its favorable chemical

characteristics for rhizobial growth, make filter mud an

excellent prospect for a Rhizobium inoculant carrier

material for St. Kitts.















CHAPTER 5
BEAN FIELDINOCULATION TRIALS

Introduction

Field inoculation trials were conducted in order to

determine if indigenous Rhizobium populations, or lack

thereof, pose a constraint to bean production in St. Kitts.

Since dry bean production was virtually non-existent on the

island, no established cultivars were present for use in

inoculation studies. Therefore, inoculation trials

included several cultivars from the Caribbean region and

the U.S. to assess cultivar and inoculation treatment

interactions and to evaluate and compare the agronomic

performance of these cultivars. The results of these

trials should provide useful information for selecting

cultivars for bean production in St. Kitts.

Three field inoculation bean trials were completed.

The specific objectives of these trials were

1) to evaluate nodulation and growth of beans as

affected by Rhizobium inoculation and N applica-

tion;

2) to compare growth and nodulation responses of

several bean cultivars to Rhizobium inoculation

and N application; and









3) to compare the agronomic performance of several

cultivars under rainfed and irrigated conditions.

In each of the three experiments, three basic inoculation

treatments were evaluated. These three treatments, which

form the core of a standard Rhizobium inoculation trial,

included uninoculated and N-fertilized controls and

Rhizobium inoculation.

In the first experiment conducted at the CARDI Research

Station in 1983, seed and soil methods of inoculation were

tested along with the uninoculated and N-fertilized

controls. Five different cultivars obtained from

researchers in the U.S. and the Caribbean were evaluated.

Selected cultivars from the first trial, plus an

additional cultivar obtained locally, were evaluated in the

second and third trials. The second and third trials

differed only in that the second trial was conducted on-

farm under rainfed conditions while the third experiment

was conducted with irrigation at the CARDI Research

Station.


Materials and Methods

1983 Trial

The first trial was conducted in the southeast section

of the CARDI Research Station. The site had no known

history of bean inoculant use. A strip-split-plot design

with four replications was employed. Two rows of each of

five cultivars were stripped across four inoculation

treatments which served as main plots. Two of the







78

cultivars were obtained from Dr. Adet Thomas, an agronomist

working at that time for CARDI in Jamaica; the other three

cultivars were obtained from Dr. Fred Bliss of the

University of Wisconsin. A list of the five cultivars and

relevant information is found in Table 5-1.

The four Rhizobium inoculation treatments imposed upon

the five cultivars were

(1) uninoculated control,

(2) N, as urea, applied at 100 kg ha-1

(3) inoculated via a seed-applied powdered inoculant,

(4) inoculated via a soil-applied granular inoculant.



Table 5-1. Bean cultivars evaluated in the 1983 Rhizobium
inoculation trial.


Name Seed type Growth habit Source


Round Red medium semi-vining CARDI
red Jamaica


Miss Kelly medium semi-vining CARDI
red pinto Jamaica


California large non-vining Univ. of
Red Kidney red kidney Wisconsin


Porillo small semi-vining Univ. of
Sintetico black Wisconsin
(21-57)


Sanilac medium semi-vining Univ. of
(24-18) white navy Wisconsin









The Rhizobium inoculants used in this experiment were

commercial products obtained from Dr. Stewart Smith of the

NITRAGIN Company, Milwaukee, Wisconsin. Inoculants con-

tained the two R. phaseoli strains Nitl27kl2b and

Nitl27k44.

A complete micronutrient mix, TEM300, was broadcasted

at the rate of 50 kg ha-1 and incorporated 1 week prior to

planting. TEM300 contained 3% B, 3% Cu, 18% Fe, 7% Zn,

7.5% Mn, and 0.7% Mo. No P or K fertilizer was applied due

to high soil test levels of these nutrients (Table 5-2).

Nitrogen, as urea, was applied to appropriate plots in

split applications: 50 kg ha-1 were broadcast at planting

and scratched into the surface by raking, and 50 kg ha-1

were sidedressed at bloom (35 days) in a wide band between

rows and incorporated with an inter-row cultivation.





Table 5-2. Chemical analyses of soil prior to planting of
field inoculation trials at CARDI in August,
1983.


Mehlich II extractablel

pH$Q
Ca Mg K P Mn Zn Cu


- - - - - mg kg------- -

6.0 2000 500 410 260 7 10 0.4


t 0.2 M NH4C1 + 0.2 M HOAc + 0.015 M NH4F + 0.012 M HC1;
pH 2.5.
t$ 1:2 soil:water (v:v).









Beans were planted on 18 Aug. 1983. Seed inoculation

was accomplished as described by Somasegaran et al.,

(1982). Batches of seed (200 g) were first moistened with

3 to 4 mL of neutralized 40% gum arabic sticker and then

coated with 10 g of powdered peat inoculant by gently

shaking in a plastic bag. The granular inoculant was

applied by hand directly to the bottom of the 3-4 cm deep

planting furrow at the rate of 50 g per 15 m of row

immediately before the seeds were sown.

Soil temperature at planting was measured at two

different depths with a standard glass-mercury thermometer.

The temperature was taken by inserting the thermometer 3 cm

deep into the soil and then 15 cm deep after removing the

top 10 cm of soil. Four measurements were taken per

replication.

At the start of the experiment, the only possibility

for irrigation was to run several garden hoses down from

the station building to the plots. Although this method

was used to water the area before planting, it was

unacceptable once the field was planted. After several

weeks of dry weather, a lateral line was cut into the main

water line to permit a permanent irrigation system to be

installed in the plots. Therefore, from bloom to harvest,

irrigation was provided as needed.

A plant harvest was made at first bloom, 32 and 33 DAP.

Ten plants were excavated from each plot for nodule counts

and top dry weight determinations. Nodules were counted in

the field without removing them from the roots. Leaf







81

samples, composed of 10-20 leaves per plot, were taken at

the same time. Leaf samples were dried, ground, and

analyzed for N. Beans were harvested 11-15 November. Due

to poor and uneven stands, seed yields were estimated by

harvesting ten plants per plot from sections of row with

near equal within-row spacing.


1985 and 1986 Trials

Two bean inoculation trials were conducted. One

experiment was conducted on a local farm owned by Dodridge

"Brotherman" Huggins while the other was conducted at the

CARDI Research Station. The experimental design used in

each of these two trials was a randomized complete block

with four replications. Four cultivars including Miss

Kelly, Round Red, and Red Kidney from the first experiment

and a locally available pink bean called Sutter Pink were

each evaluated under three inoculation treatments

1) uninoculated control,

2) N, as urea, applied at 100 kg kg-,

3) Rhizobium inoculated.

Unlike the first trial, inoculants used in these trials

were locally prepared. At Huggins, peat was used as the

inoculant carrier while filter mud was used at CARDI.

Two commercial strains of Rhizobium phaseoli,

Nitl27K12b and Nitl27K44, were obtained from Dr. Stewart

Smith of the NITRAGIN CO. for use in this experiment. The

strains were cultured separately before addition to the

carrier to make a double-strain inoculant. Other than the







82

use of two strains, inoculant preparation was identical to

that detailed in the inoculant carrier study (p. 66). The

broth cultures were checked for purity by the drop-plate

technique and authenticated by pipetting 1 mL of broth on

to Miss Kelly seedlings grown in growth pouches. No counts

were made on the final inoculant product.

Composite soil samples from both sites were collected

for quantification of R. phaseoli by MPN methodology. For

the MPN tests, 100 g of moist (12% and 15% dwb for Huggins

and CARDI soils, respectively) soil were shaken with 900 mL

of distilled water for 10 min. Subsequently, four-fold

dilutions in water were made from which 1 mL aliquots were

pipetted on to seedlings of cv. Miss Kelly in quadruplicate

growth pouches. Growth pouches were heat-sealed so as to

provide two compartments per pouch. The MPN was determined

using tables from Vincent (1970).

From the same soil composites, Mehlich I-extractable

(0.05 M HC1 + 0.0125 M H2SO4) Ca, Mg, K and P were deter-

mined. Soil pH was determined in a 1:2 water suspension.

Both total Kjeldahl N (TKN) and mineral N (NH4+, N03-) were

determined on a finely ground (minus no. 100 mesh sieve)

subsample by the standard procedures of Bremmer and

Mulvaney (1982). Soil TKN was analyzed with salicylic

acid-thiosulfate methodology to include nitrates. Mineral

N was extracted with 1 M KC1 and distilled with MgO-Devarda

alloy to include nitrates and nitrites.







83

Individual plots were 7 m long by 90 cm wide flat beds

of three rows. There were 60 cm between beds giving a

total plot width of 1.2 m and a plot area of 8.4 m2.

A broadcast application of fertilizer consisting of 50

kg P ha-1 as OSP, 80 kg K ha-1 as KC1 and 40 kg TEM300 ha-1

of TEM300, a commercial micronutrient mix (see p. 79), was

incorporated 1 week prior to planting. No herbicides were

used prior to or at planting. For the 100 kg N ha-1

treatment at Huggins, 50 kg ha-1 were mixed with the

preplant P and K; the other 50 kg ha-1 were applied at

flowering. At CARDI, 75 kg N ha-I were applied 6 days

after planting by broadcasting the urea between the three

rows and lightly raking it into the soil; the remaining 25

kg/ha were applied at flowering in the middles and incor-

porated during a weed cultivation. Irrigation was applied

to help prevent volatilization losses.

Cultivars were planted 6-8 Sept. 1985 at Huggins and 1-

2 Feb. 1986 at CARDI. Approximately 2 L of water per plot

were delivered by gravity feed to the bottom of the furrow

prior to sowing the seed. For the inoculated treatment,

100 g of inoculant were suspended in the 2 L of water. The

seeds were planted by hand at a 6-8 cm within-row spacing.

Plants were later thinned to a within-row spacing of 12-15

cm. Overhead spray mist irrigation lines were installed at

the CARDI site.

Irrigation was supplied at CARDI every 3 to 6 days as

needed throughout the cropping period. Weeds at both

locations were controlled by a slice-hoe cultivator between









rows and by hand pulling within the row. Insecticide/

fungicide mixtures were applied on an approximately

biweekly basis for pest control.

Leaf samples were collected from each plot on 6 Oct. at

Huggins and on 2 March at CARDI. Visual ratings were taken

on 6 Oct. and 28 Oct. at Huggins and on 2 March and 26

March at CARDI. Ratings were based on a 0 to 4 scale with

0 being very chlorotic and 4 being dark green. Ten plants

were excavated from each plot on 6-8 Oct. at Huggins and on

7-9 March at CARDI; three plants were chosen from each of

the two outside rows and four from the inside row.

Nodulation was rated at Huggins while at CARDI nodules were

removed, washed, counted, dried, and weighed. The rating

system used at Huggins was a modification of that used by

INTSOY (1981) for soybean. In this scoring system,

consideration was given to the number of nodules observed

on the main tap root and on lateral roots: 0=no nodules;

l=few (<10) on lateral roots; 2=many (>10) on lateral

roots; 3=few (<5) large nodules on tap root and many on

laterals; 4=many (>5) large tap root nodules and many on

laterals. Only mature nodules as determined by the

characteristic rough surface texture were counted. The

fresh weight of the ten whole plants per plot was also

determined.

Plots were harvested on 11-19 Nov. at Huggins and 14-

18 April at CARDI by pulling plants from 5 m of the three-

row bed. At Huggins, pods were pulled off the plants and

taken to CARDI for drying on screens; at CARDI, the plants







85

were pulled, bundled, and left to dry outside until ready

for threshing. Pods containing at least one seed were

counted in two 0.5 m2 zones in the harvest area marked for

pod density determinations. From the pods collected, the

number of seeds per 50 randomly selected pods was

determined. After shelling, the seeds were cleaned and

unacceptable seeds were discarded. From these, 250 seeds

were counted out for moisture content and seed size (g 100

seed-1) determinations. Plot yields were converted to kg

ha-1 corrected to 13% moisture.


Results and Discussion

1983 Trial

Plant stands and early growth for the 1983 bean

inoculation trial were unsatisfactory due to a combination

of factors including bird damage, damping-off disease, and

a lesser cornstalk borer (Elasmopalpus lignosellus)

infestation. Birds reduced stands by pulling out emerging

cotyledons. The damping-off disease was evident by

reddish-brown root rot typical of the damage caused by

Rhizoctonia complex or Pythium. The incorporation of fresh

plant material 1 week prior to planting in combination with

a heavy irrigation before planting may have enhanced this

problem. Seeds were not treated with fungicides in order

to minimize potential toxic effects on rhizobia (Graham et

al., 1980). An infestation of lesser cornstalk borer made

worse by the dry, hot weather appeared 10 to 14 days after

emergence and caused further reductions in stands.









Nodulation and growth responses of beans to Rhizobium

inoculation and N application in 1983 varied among the

different cultivars (Table 5-3). Nodule number, which is

an indicator of inoculation success, was increased by

Rhizobium inoculation for each cultivar tested. Nodulation

was greatest for cvs. Porillo Sintetico (21-57) and Miss

Kelly and least for Round Red. Limited nodulation of Round

Red even with inoculation may have been confounded by a

relatively high incidence of damping-off disease observed

for this cultivar. Orellena et al. (1976) found that root

rot reduced both nodule weight per plant and N fixed after

60 days.

Although inoculation was successful in increasing

nodulation, seed yields were increased only for Porillo

Sintetico (21-57) and Miss Kelly. Rhizobium inoculation

increased yield 65% (500 vs 300 g 10 plant-1) and 18% (540

vs 450 g 10 plant-1) for cvs. Porillo Sintetico and Miss

Kelly, respectively. These increases were approximately

equivalent to increases observed from the application of

100 kg N ha-1. For Porillo Sintetico (21-57), yield

increases from inoculation were not reflected in plant dry

weight or leaf N conc at bloom indicating that early growth

and N composition were not enhanced by increased nodula-

tion. While plant dry weight of Miss Kelly was increased

by inoculation, no response in leaf N conc was observed.

No differences in nodulation or growth of beans were

observed between the two methods of inoculation. The

application of granular peat inoculant has been shown to









Table 5-3.


Nodulation and growth responses of bean
cultivars to Rhizobium inoculation and N
application at CARDI, 1983.4


Nodule Plant Leaf Seed
Cultivar Treatment number dry wt N yield


no. pl-1 g pl-1 dag kg-1 g pl-1

Porillo Control 25 5.1 4.92 30
(21-57) N## 8 6.5* 5.15 50*
Inoc (seed) 109 4.8 4.88 51*
Inoc (soil) 86 5.3 4.95 48*

Miss Kelly Control 31 5.0 4.78 45
N 10 6.6* 5.10 58*
Inoc (seed) 62 6.2* 4.97 52*
Inoc (soil) 60 6.3* 4.96 55*

Round Red Control 10 5.2 3.67 14
N 5 6.3* 4.68* 36*
Inoc (seed) 18 5.5 3.46 16
Inoc (soil) 15 5.1 3.69 15

Red Kidney Control 28 4.9 4.68 34
N 16 9.6* 5.68* 65*
Inoc (seed) 45 6.1* 4.57 33
Inoc (soil) 41 5.9 4.88 31

Sanilac Control 15 6.1 4.28 31
(21-18) N 3 7.1 5.23* 44*
Inoc (seed) 45 7.1 4.41 33
Inoc (soil) 44 6.3 4.23 35


LSD.05 NS 1.04 0.338 5.8

CV (%) 53.8 8.8 3.6 8.0


4 Means represent the average of 4 replications.
44 Nitrogen, as urea, applied at 100 kg N ha-.
Means are significantly different than the uninoculated
control for a particular cultivar according to
LSD0.05. Due to non-homogeneity of variance, the LSD
test for the nodule number parameter was performed
only between inoculated treatments (i.e., seed versus
soil inoculation).









increase nodulation of legumes under conditions where a

greater number of rhizobia need to be applied (Burton,

1979). Due to the sensitivity of most rhizobia to heat

(Norris and Date, 1976), large inoculum rates may be

advantageous when planting under hot soil conditions. soil

temperature at planting ranged from 34-390C (94-1030F) at

the surface 3 cm and 29-320C (85-900F) at the 15 cm depth.

Soil inoculation has also proven beneficial when

chemically-treated seed are used (Graham et al., 1980). As

previously noted, bean seeds were untreated in these

experiments. Also, there is some concern that by raising

their seed coats aboveground during emergence, legumes with

epigeal emergence (most major grain legumes) limit the

number of rhizobia that are present in the root zone for

root infection (Brockwell, 1977). By placing the inoculant

at the bottom of the furrow, not only can one apply greater

numbers of rhizobia per seed, but these rhizobia are placed

in the root zone where they are needed. Despite inoculant

application rates approximately seven times higher with

granular inoculant than seed inoculant (3.3 vs 0.5 g

inoculant m-1 row), no differences in nodulation or growth

were obtained at CARDI in 1983 relative to inoculant form.

Despite increased nodulation, seed yields of cvs. Round

Red, Red Kidney, and Sanilac (21-18) were not increased by

Rhizobium inoculation. However, the application of 100 kg

N ha-1 increased seed yield 151, 91, and 39% for these same

cultivars, respectively. Seed yields of each of the these

cultivars was increased by N but not inoculation,







89

indicating that these cultivars depended more upon mineral

N than symbiotic N relative to the Porillo Sintetico and

Miss Kelly. Alternatively, but less likely, these

cultivars were not compatible with inoculant or commercial

strains of rhizobia.

Plants under N stress will often exhibit low leaf N

concentrations. A leaf N conc at bloom of 3 dag kg-1 has

been commonly used as a critical value below which bean

yields will be greatly reduced (Geraldson et al., 1973).

Leaf N conc of all samples were above this critical value.

Leaf N conc was increased by N application but not by

inoculation. The dramatic effect of N application in

increasing leaf N conc and yields of the three cultivars

which did not respond to inoculation appeared to be due to

poor early growth. These cultivars were not as hardy as

Porillo Sintetico and Miss Kelly but seemed to recover well

once N and irrigation were applied.

Conclusions drawn from this initial experience of

growing beans in St. Kitts were limited by the inadequate

stands and poor early growth. Results indicated that two

out of the five cultivars tested benefited from Rhizobium

inoculation. Inoculant strains appeared to be more effec-

tive than indigenous strains for Porillo Sintetico (21-57)

while Miss Kelly yielded relatively well without N applica-

tion or inoculation. Although an absence of a growth

increase from inoculation for the other three cultivars may

have been confounded by early plant stress conditions,









these cultivars appeared to be more responsive to ferti-

lizer N than Porillo Sintetico (21-57) and Miss Kelly.


1985 and 1986 Trials

Trials conducted in 1985 at Huggins and in 1986 at the

CARDI Research Station were much more successful than the

1983 trial relative to pest and environmental problems. A

bacterial blight which appeared during both experiments was

the only unfavorable pest or disease problem encountered.

When leaf sections were placed on potato dextrose agar,

yellow colonies developed which are characteristic of

common bacterial blight (Xanthomonas phaseoli). Dr. Osburt

Liburd, a plant pathologist and Program Director for St.

Kitts, aided in this identification. Excellent plant

stands were achieved for both sites and weeds and insects

were effectively controlled.

When making the filter mud inoculant (CARDI site), the

broth culture of strain Nitl27K12b was contaminated with

large Gram-positive rods at 104 cells per mL broth; the

Rhizobium count for the same broth was 109 cells per mL of

broth. Although this contamination was unwanted, the

preparation was not repeated since the rhizobia counts were

high and the inoculants were not stored for a long time

before use. Also, very high application rates were used

(4.8 g m-1 of row versus standard rates of 0.03 and 0.5 g

m-1 of row for seed and soil inoculation, respectively).

No significant contamination was found in the other broth









cultures. All broth cultures produced nodules on Miss

Kelly grown in growth pouches.

Characteristics of Huggins and CARDI sites are provided

in Table 5-4. Extractable nutrients at both sites were

high. The high K value for Huggins was a result of exces-

sive potash applications by Mr. Huggins. Mineral N





Table 5-4. Characteristics of Huggins and CARDI sites
1985 and 1986 bean inoculation trials were
conducted.


Huggins CARDI


Altitude (m) 200 50
Soil texture sandy loam loam
Soil pH (1:2 H20) 6.3 7.1
Mehlich I-extractable4: Ca 1050 1880
(mg kg-1) Mg 176 420
K 400 110
P 42 68
Total Nt$ (g kg-1) 1.15 1.44
Mineral NP (mg kg-1) 15 21
MPN bean rhizobia (no. g-l) 320 1000


$ (0.05 M HC1 + 0.0125 M H2S04)
tt Including nitrates
3 KC1 extractable using MgO-Devarda alloy methodology

was higher than expected at both sites. Assuming 2 x 106

kg soil ha-1, 30 and 42 kg N ha-1 were available at

planting without N fertilization. The higher soil pH of

7.1 at CARDI reflected the impact that irrigation water had

at that site. The soil pH of 6.0 for the 1983 CARDI trial

was lower because irrigation had not been available on that


section of the station.







92

Both sites contained relatively high populations of R.

phaseoli. According to Date (1982), soils with less than

100 rhizobia g-1 of soil would be considered to have low

populations of rhizobia and soils with more than 104

rhizobia g-l of soil would be considered to have high

populations of rhizobia. Weaver and Frederick (1974)

reported that soybean nodulation was not increased by

inoculation in soils containing greater than 1000 B.

japonicum g-l. In the previously described pot study,

inoculation increased dry weight yield of Miss Kelly beans

grown in Huggins soil despite the fact that uninoculated

controls nodulated well with indigenous rhizobia. In fact,

the 63% increase in plant dry weight yield from inoculation

was the greatest response of all 35 soils tested. During

the same study, no response to inoculation was observed for

the CARDI soil in which uninoculated controls were also

well-nodulated.

Rainfall recorded in cm (inches) at the Fountain Estate

near Huggins was 11.2 (4.4) in Sept., 17.8 (7.0) in Oct.,

and 12.7 (5.0) in Nov. A 3-week period of hot, dry

conditions was experienced in the middle of October. At

CARDI, rainfall in cm (inches) recorded at nearby estates

was 1.8 (0.7) in Feb., 3.6 (1.4) in March, 9.4 (3.7) in

April, and 10.7 (4.2) in May. Irrigation was used fre-

quently throughout February and March.









Cultivar performance

Selected characteristics of the four dry bean cultivars

included in the 1985 and 1986 trials are presented in

Table 5-5. Cultivars Miss Kelly and Round Red are the most

popular dry bean cultivars in Jamaica (Adet Thomas, 1980)

and Miss Kelly has performed well against improved red-

seeded lines in variety trials (CIAT, 1983). Although

results with Round Red were poor during the first trial, it

was included in subsequent trials because results from the

first trial were inconclusive and because local agricul-

turalists and consumers indicated that the seed size and

color gave it a much greater chance for local acceptance

than other colored beans. Also, despite having lower

yields-in the 1983 trial, seeds of Round Red were of high

quality, more noticeably so than the other cultivars.

During soil sampling trips around the island, several

farmers were observed to be growing kidney beans and pink

beans which they had purchased from local food stores.

Since these beans were being grown at least to some extent

on farms, their inclusion in the inoculation trials would

have some immediate relevance. Seeds of these two bean

types were purchased in St. Kitts and found to have accep-

table germination. A complaint of several farmers was that

germination of these store-bought beans was quite variable

from bag to bag (imported in 50-kg bags). Farmers normally

will plant just a few seed to test seed viability before

planting a whole plot. The kidney bean was imported from

Stockton District Bean Growers (P.O. Box 654, Linden, CA




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