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1 EVAL UATION OF ISARIA FUMOSOROSEA FOR CONTROL OF THE A SIAN CITRUS PSYLLID, DIAPHORINA CITRI KUWAYAMA (HEMIPTERA: PSYLLIDAE) By KAREN MARIE PALANUK STAUDERMAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012
2 2012 Karen Marie Palanuk Stauderman
3 To my husband, Harry James and daughter, Lynn Marie Stauderman
4 ACKNOWLEDGMENTS I woul d like to thank my advisor and chair of my graduate committee, Dr. Steven Arthurs for his professional advice, scientific guidance and financial support. I also thank the other members of my graduate committee, Dr. Robert Stamps and Dr. Lance Osborne for their contributions to my research proposal, preparing my qualifying examination and reviewing this thesis. I acknowledge Mary Brennan and Robert Leckel for their contributions involving insect rearing and technical assistance in the greenhouse. I want to thank my family for their constant encouragement d uring my graduate experience. Finally, I wish to acknowledge my husband, Harry J. Stauderman, for his dedication, patience, and unwavering support.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 Literature Review ................................ ................................ ................................ .... 11 Citrus Greenin g Disease (HLB) ................................ ................................ .............. 11 Description ................................ ................................ ................................ ....... 11 Causal Agent ................................ ................................ ................................ .... 12 Asian Citrus P syllid ................................ ................................ ................................ 13 Description and Morphology ................................ ................................ ............. 13 Feeding and Reproduction ................................ ................................ ............... 14 Economic Impact ................................ ................................ .............................. 15 Management of D. citri in Citrus Groves ................................ ................................ 16 Chemical Control ................................ ................................ .............................. 16 Integrated Crop Management ................................ ................................ ........... 18 Entomopathogenic Fungi for Control of D. citri ................................ ....................... 19 2 MATERIALS AND METHODS ................................ ................................ ................ 24 Objective 1. Determine the Pathogenicity and Virulence of I. fumosorosea Strains to D. citri ................................ ................................ ................................ .. 25 Fungal Isolates ................................ ................................ ................................ 25 Statistical Analysis ................................ ................................ ............................ 27 Objective 2. Assessment of D. citri Mortality Using I. fumosorosea with and without Emulsifiable Oils in Greenhouse Tests. ................................ ................... 28 Bioassay Procedure ................................ ................................ ......................... 28 Statistical Analysis ................................ ................................ ............................ 32 3 RESUL TS ................................ ................................ ................................ ............... 33 Stages of Development of I. fumosorosea In Vitro and in D. citri ............................ 33 Survival and Mycosis of D. citri in Laboratory Bioassa ys ................................ ........ 33 Evaluation of Foliar Sprays of I. fumosorosea and Emulsifiable Oils against D. citri Infestations on Citrus under Greenhouse Conditions ................................ .... 34 4 DISCUSSION ................................ ................................ ................................ ......... 46
6 5 CONCLUSIONS ................................ ................................ ................................ ..... 53 LITERATURE CITED ................................ ................................ ................................ .... 54 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 64
7 LIST OF TABLES Table page 3 1 Average survival times (days) of adult D. citri exposed to 4 strains/formulations of Isari a fumosorosea at concentrations between 10 3 and 10 9 spores per ml. ................................ ................................ ........................ 40 3 2 Estimates of the median lethal concentration (spores/ml) of three entomopathogenic fungi applied against adult D. citri on Marsh grapefruit leaf disc assays at 7 days post inoculation. ................................ ............................... 41 3 3 Number of infested terminals and ACP recorded on orange jessamine plants following treatment applications in the fall 20 10 test. ................................ .......... 42 3 4 Number of infested terminals and ACP recorded on Benton citrange plants following different treatments (summer 2011 test). ................................ ............. 43 3 5 Number of infested terminals and ACP recorded on Benton citrange plants following different treatments (fall 2011 test) ................................ ...................... 44 3 6 Number of ACP on orange jessamine plants as determ ined by a final destructive count (fall 2010 test) ................................ ................................ ......... 45
8 LIST OF FIGURES Figure page 1 1 Symptoms of HLB in citrus foliage and fruit, and the vector D. citri ................... 22 1 2 Schematic showing how entomopathogenic fungi infect their insect hosts through their cuticles. ................................ ................................ ......................... 23 3 1 Photographs s howing stages of development of I. fumosorosea in D. citri .. ....... 36 3 2 Proportional survival of adult D. citri following exposure to citrus leaves treated with different formulations of Isaria fum osorosea ................................ ... 37 3 3 Proportion mycosis (sporulation) of adult D. citri following exposure to Marsh grapefruit leaves treated with different formulations of Isaria fumosorosea ....... 38 3 4 Cladosporium sp. contamination observed on Benton citrange in greenhouse cages. ................................ ................................ ................................ ................. 39
9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EVAL UATION OF ISARIA FUMOSOROSEA FOR CONTROL OF THE A SIAN CITRUS PSYLLID, DIAPHORINA CITRI KUWAYAMA (HEMIPTERA: PSYLLIDAE) By Karen Marie Palanuk Stauderman May 2012 Chair: Steven P. Arthurs Major: Entomology and Nematology The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a serious pest of citrus and the vector of citrus greening disease, also called h uanglongbing (HLB), in Florida. Alternative low risk pesticides are needed given the risks of over using broad spectrum chemical pesticides and the need to conserve beneficial arthropods in citrus production. This study evaluates insect specific (entomopathogenic) fungi for the control of D. citri A bioassay protocol was developed to evaluate strains of the entomopathogenic fungus, Isaria fumosorosea Wize, against D. citri Up to 100% of adult psyllids were killed at concentrations between 10 6 and 10 7 blastospores/ml after 12 days with derived LC 50 values (at 7 days) of 1.3710 5 (ARSEF 3581), 2.0310 6 ( Apopka 97), 1.36 10 5 (FE 9901), 1.47 10 7 blastospores/ml and 1.4710 7 for a conidial formulation of Apopka 97. Average survival times were dosage dependent for all strains, i.e. betwe en 10.2 days at 10 3 blastospores /ml and 3.1 days at 10 9 blastospores/ml. Rates of symptomatic fungal mycosis observed in dead psyllids were also concentration dependent, with up to 100% sporulation observed at concentrations of 10 8 blastospores or higher b ut declined at lower concentrations Based on the laboratory screening, the Apopka 97 strain (commercially available as a
10 bioinsecticide called PFR 97) was tested against D. citri infestations in citrus plants under greenhouse conditions. Half of the formu lations included an em ulsifiable vegetable oil at 2.5 percent vol/vol that was hypothesized to improve fungal efficacy, such as through improved deposition or germination on the insect cuticle. Fungal treatments at label rates reduced psyllid populations b y approximately 50 percent over four weeks. The combination of PFR 97 and the emulsifiable oil did not increase ACP mortality compared with either agent alone. Subsequent greenhouse tests conducted under humid conditions were hampered by natural disseminat ion of the fungus to untreated psyllid populations, suggesting that the fungus is spread easily by wind or other factors. The insecticide imidacloprid applied as a drench was highly effective, killing 100% of psyllids within 3 weeks. This study demonstrate d the potential of entomopathogenic fungi for environmentally safe control of D. citri although it may not be so effective as chemical insecticides.
11 CHAPTER 1 INTRODUCTION Literature Review A serious disease currently affecting the Florida citrus industry is Huanglongbing or greening disease. A problematic aspect of the disease is that it is transmitted by an insect vector, t he Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae) The objective of t his chapter is to present an over view of the disease and its cause, of the vector with respect to its biology, host range and pest status, of the control and integrated pest management of D. citri and of how the control and management has changed since HLB was discovered in Florida. Alte rnative low risk pesticides are needed both because of the risks of over using broad spectrum chemical pesticides and the need to conserve beneficial arthropods (parasitic wasps, predatory bugs, spiders, etc) in the groves. Citrus Greening Disease (HLB) De scription 81). The earliest record of HLB was in Shinchiku, Taiwan in 1907 (Kuwayama 1908) and it has successfully invaded citru s producing areas throughout the world. Citrus fruit from infected trees do not produce color but remain green on the tree. Early symptoms of HLB include blotchy mottled leaves with a yellowing of a single sector of the tree canopy ( Fig 1 1 ). By contrast, nutritional deficiencies occur symmetrically along the leaf veins. Later symptoms include twig and limb dieback, sparse, stunted leaves that point upward and premature fruit drop. Infected citrus trees may only live 5 8 years and produce irregular
12 shaped, bitter, unmarketable fruit (Bov 2006; Halbert and Manjunath 2004). According to the U.S. Department of Agriculture, HLB is a growing threat to citrus production in Texas, California and Florida (Santa Ana 2012; USDA 2011b). There are at least three recog nized forms of phloem limited gram negative bacteria that are causal agents of HLB worldwide (Bov 2006). Candidatus Liberibacteria asiaticus is the most widespread and severest of the three. It thrives under low humidity in both cool and warm temperatures (heat tolerant up to 35 C). Vectored by D. citri HLB occurs throughout Asia, India and neighboring islands, Saudia Arabian peninsula, Brazil, Louisiana and Florida (Garnier and Bov 1993). The African form, Candidatus Liberibacter africanus is a milder form of the disease that is restricted to south of the Sahara in Africa, is vectored by the African citrus psyllid Trioza erytreae Del Guerico ( Magomere et al. 2009; Van de Berg 1990) and is considered heat sensitive (Bov 2006; Le Roux et al 2006). Mor e recently, in 2004, a third form was discovered in Brazil, Candidatus Liberibacter americanus (Coletta Filho et al 2004; Teixeira et al 2004). This form is only found in Brazil where it is vectored by D. citri Causal Agent The Candidatus Liberibacter a siaticus bacterium is either spherical or rod shaped, gram negative and is found in phloem cells. These bacteria average 930 nm in length and 410 nm in width. According to Shokrollah et al (2010), the ce ll walls are irregular with varying thickness. It is believed that the bacteria damages cell walls by penetrating and moving throughout the cells. The bacteria have not been cultured in media and, therefore, there is insufficient information on the movement of the pathogen in citrus plants (Shokrollah et al 2010).
13 HLB is transferred to other plants primarily by piercing sucking insects. Psyllids are the main vector of this disease. The disease is also known to be graft transmitted, transferred by humans in movement of host material, transmitted experim entally via dodder (Gottwald 2010), and possibly seedborne (Sullivan and Zink 2007). Asian Citrus Psyllid Description and Morphology D. citri is endemic to Asia and known to have a wide host range within the plant family Rutaceae, specifically citrus and related species including orange jessamine Murraya paniculata L. Jack (Halbert and Manjunath 2004 ; Swingle and Reece 1967 ). D. citri was first detected in Florida in 1998 (Knapp et al 2006) and has since spread throughout the state (Childers and Rogers 2 005; FDACS 2008; Qureshi and Stansly 2007b; Tsai et al 2000). D. citri has also been found in Texas (French et al 2001), Louisiana, Alabama, Georgia, Mississippi, South Carolina, California, Puerto Rico and Guam ( Hummel and Ferrin, 2010; USDA 2008 ; USDA 2011b ) and a ll of the islands of Hawaii (Conant et al 2009). How it arrived in Florida and exactly where it came from is not known ; however discount garden centers and retail nurseries may have helped distribute psyllids and plants carrying the HLB pathogen in Flori da (Manjunath et al 2008). Mead (1977) described D. citri as ranging in length from 2 4 mm N ymphs are yellow in color. D. citri can be distinguished from six other similar species ba sed upon the pattern of marks in the forewings (Halbert and Manjunath 2004). Eggs of D. citri are oval shaped and about 0.3 mm long ; as they mature ; they turn dark yellow to orange. The eggs hatch after 3 4 days and the psyllid develops through 5 instars. Nymphs continue to
14 mature for 12 to 14 days, although this period has been shown to vary with environmental conditions of temperature and humidity (Liu and Tsai 2000; McFarland and Hoy 2001) S ome D. citri may survive moderate freeze events in citrus grove s and adults may become cold acclimated during exposure to cooler temperatures during the winter (Hall et al 2011). Both nymphs and adults produce white waxy secretions containing honeydew that accumulates on the leaf surfaces. Honeydew promotes the growt h of sooty mold fungi, which is unsightly in ornamental settings and can reduce effective leaf area for photosynthesis (Browning et al 2009). Feeding and Reproduction The two factors most important for D. citri feeding and reproduction are the presence o f new citrus flush and warm temperature s Ideal temperature s in the range from 20 30C correspond directly with the abundance of psyllids in the field (Tsai et al 2002). These environmental conditions are also correlated with shoot flush cycles in citrus. Extended temperatures above 32 C will decrease the female psyllid lifespan and egg production. Tsai et al (2002) reported that Florida weather conditions decreased psyllid populations during mid summer months compared to late spring and early fall. Addit ionally, they noted that orange jessamine, Murraya paniculata L. (an ornamental shrub) serves as an alternate host to the Asian citrus psyllid. The flushing pattern of the citrus relative is continuous in southern Florida and this enables psyllid densitie s to peak in May, August, and October through December (Tsai et al 2002). When young, tender unexpanded leaves are present, adult psyllid commonly aggregate on this new flush where they feed and mate. With piercing sucking mouthparts, their feeding result s in deformed, twisted leaves and reduc ed shoot length s and possible transmission of HLB (Michaud 2004). Psyllid also siphon large amounts of
15 plant phloem and excrete the excess as honeydew and wax at the feeding site (Aubert 1987; Triplehorn and Johnson 2005). After mating, the female psyllid must feed on young flush in order to produce mature eggs. Gravid females eventually develop a yellowish to orange abdomen but recent studies show that both sexes can reach reproductive maturity by 2 3 d post eclosio n (hatching) before this color change occurs (Wenninger and Hall 2007 ; Wenninger et al 2009) Eggs are inserted into the leaf tissue inside the folds of the new flush of leaves The life span ranges from 30 50 days depending upon humidity, temperatures an d host plant (Liu and Tsai 2000; McFarland and Hoy 2001). Under optimum conditions more than 10 generations of psyllids can be produced per year (McFarland and Hoy 2001). Although D. citri damages plants directly through its feeding activities, the most se rious concern of D. citri in Florida, and world wide, is its ability to vector the phloem limited bacterium Candidatus Liberibacter asiaticus that causes h uanglongbing (HLB), also known as citrus greening disease (Hung et al 2004; Manjunath et al 2008). HLB is transmitted by the probing action of psyllids during feeding. As little as 30 minutes of feeding has been known to transmit the bacterial pathogen (Bov 2006). Once the psyllid has acquir ed the bacteria, there is a latent period that varies from 7 2 5 days before it can transmit the pathogen which is thought to occur through salivary secretions. Adult psyllid can live for 1 2 months and once they have acquired the HLB bacterium they carry it for life, transmitting it to additional citrus during feedi ng (Bov 2006). Economic Impact When D. citri first arrived in Florida, it was not considered by some to be a serious pest per se Healthy mature citrus trees could withstand significant damage to young
16 growing shoots, although young plants may succumb dur ing high populations of the psyllid (Michaud 2004). Additionally, D. citri was a pest that was easily controlled b y the routine insecticidal sprays used by most growers. However, when HLB was detected in south Florida in 2005 on two homeowner trees (FDACS 2005) and spread to numerous (Hodges and Spreen 2012) Citrus acreage in the state has declined to its lowest level i n years. C itrus acreage for the 2010 2011 season decreased 6% from the previous season to 541,000 acres, which is considerable reduction from the peak of 940,000 acres that existed during the 19 90s (USDA 2011c) Elimination and removal of trees because of residential and industrial development, poor quality soils, nematodes, infect ions of citrus greening or citrus canker, and abandoned groves were some of the many factors that helped contribute to the gross loss of 19,918 acres ( Mossler 201 1 ; U S D A 2011a) According to the Citrus Research & Development Foundation Inc. (CRDF 2011 ), most recently funded projects mainly fall within three areas reducing the bacteria inoculum within the tree, providing vector control strategies, and developing new rootstocks and scions for Florida citrus (CRDF 2011; Grosser et al 2011 ; USDA 2011a) Management of D. citri in Citrus Groves Chemical Control In an IPM program, a priority should be placed on natural mortality of the ACP wherever possible Currently, an infected tree with HLB cannot be cured. Consequently controlling psyllid populations with petroleum oil and foliar and systemic insecticides is currently recommended to reduce the risk of HLB infection (Childers and Rogers 2005;
17 Rae et al 1997; Rogers and Timmer 2007). In other areas around the world, the use of insecticides to control the psyllid vector has been the major emphasis of greening management strategies. For example, chemical control using pesticides was important in the battle against this pest in its nativ e range in China where 10 13 sprays of pesticide yearly (Tolley 1990) were required to rehabilitate citrus production in an area affected with HLB According to the 2 011 Florida Citrus Pest Management guide (Brlansky et al. 2011) the use of certified dis ease free trees is essential to minimize further spread of the disease Soil applied systemic insecticides provide the best protection. Currently aldicarb and imidacloprid are two active ingredients that are available to effectively control psyllids on you ng non bearing trees (Qureshi and Stansl y 2008; Rogers et al. 2011; Stamou et al. 2010). In addition, several other broad spectrum foliar insecticide applications are used in winter on bearing citrus trees. Recently, general guidelines were established fo r growers using air blast type sprayers (Hoffman et al. 2010) in the control of ACP in citrus groves. The method of low volume application is commonly used by growers in grove applications which allows them to target the pest quickly, effectively and usi ng minimal product. They were able to maximize efficiency in droplet size in order to penetrate the tree canopy. By applying pesticides very early in the spring, growers can help prevent the need for further sprays during bloom when pollinators are presen t (Childers and Rogers 2005; Rogers and Timmer 2007). Trivedi et al. have successfully isolated and characterized multiple beneficial bacterial strains from citrus roots by selecting bacteria antagonistic to Candidatus Liberibacter asiaticus These strains have the potential to enhance plant growth and suppress HLB.
18 Alternatively, cultural methods, such as removing diseased trees and planting disease free nursery stock, are also recommended as management strategies to limit the spread of HLB (Brlansky et al 2007; Rogers and Timmer 2007 ). By applying foliar nutritional applications to infected trees soon after infection, HLB symptoms may be reduced and the tree may tolerate the effects of the disease, thereby increasing tree survival and yield. Nutrient supp lementation may also induce plant resistance mechanisms that protect against the negative symptoms of infection. However, there is little evidence that these plant resistance mechanisms actually prevent diseases like HLB from occurring (Spann et al 2010). Unfortunately several problems with excessive reliance on pesticide programs have recently been documented. Recent studies have shown that D. citri populations have developed resistance to several insecticides (Tiwari et al 2011). Also, insecticides ma y be toxic to parasitoids released in citrus groves to help control D. citri (Hall and Nguyen 2010). It is known that naturally occurring predators and parasitoids play a vital role in regulating populations of D. citri and that their elimination through r eckless pesticide use could increase pest pressure and enhance the spread of HLB disease (Qureshi e t al. 2009). Therefore, integrated control programs based on conservation of natural enemies of D. citri through judicious use of insecticides and releases o f new parasitoids are needed for sustainable management of pest and disease. Integrated Crop Management The classic formula for the onset of a disease is generally characterized as a triangle. The factors include a conducive environment, the presence of the pathogen and a susceptible host. In the case of HLB, however, a vector serves as a fourth element for a disease epidemic. The objective of managing psyllid population s is to
19 manage their transmission potential in commercial citrus groves. Therefore, the best approach to optimize a sustainable program to reduce the psyllid populations is through the use of an integrated pest management (IPM) approach that relies on a variety of approaches. Reduced conventional pesticide application results in lowering production costs for growers, benefits to the environment, and reduced potential health hazards of pesticide exposure to farm workers and, if no conventional materials are used, the possibility of organic based certification. Alternative strategies will be especially important in organic production practices. Today, soil and foliar applied insecticides currently arrest ACP. This is not necessarily a long term sustainable strategy for crop management. Other alternative bio friendly measures should be pursue d as part of an overall IPM approach (NRC 2010). For example, biological control using released predators and parasitic wasps is an alternative to pesticides during the year when lower population levels are pres ent ( Stansly and Qureshi 2008; Qureshi and St ansly 2007a; 2007 b). The use of pesticides that are safe and compatible with natural control agents in the citrus groves is also important. The use of such products can provide resistan ce management tools for existing pesticides that are used by growers. A t this time, there does not appear to be a strategy that can match the insecticide approach. However, if sustainable IPM programs are introduced, they may offer the possibility of long term psyllid management with sustainable benefits (NRC 2010). Entomopat hogenic Fungi for Control of D. citri Insect killing (entomopathogenic) fungi natural pathogens of many insects, play an important role in the natural regulation of insect populations (Goettel et al 2005). Because they are safe to people, birds, fish and the majority of non target arthropods (Goettel et al 1990), entomopathogenic fungi can be used in IPM strategies for some
20 insect pests (Goettel et al 2005). The ability of their spores or conidia to pierce or puncture the host cuticle directly ( Fig 1 2 ) makes them an attractive biological control agent for phloem feeding insects (i.e. Asian citrus psyllid) because the insect does not need to consume the fungus ; surface inoculat ion is sufficient for effective infection (Avery et al. 2009, Hajek and St. Leger 1994). Entomopathogenic fungi are especially important natural and artifi cial control agents for aphids and whiteflies under warm and humid conditions ( Cabanillas and Jones 2009 ; Lacey et al 2008; Latg and Paperiok 1988; Steinkraus 2007 ). There has been relatively little research about managing psyllids using entomopathogenic fungi. U sing a detached leaf bioassay Puterka et al. (1994) found that the nymphs of pear psyll id Cacopsylla pyricola were susceptible to strains of Beauveria bassiana (Balsa mo) Vuillemin, Metarhizium anisopliae (Metschnikoff) Sorokin, M. flavoviride (Gams & Rozsypal), Isaria fumosorosea (= Paecilomyces fumosoroseus ) Wize (Hypocreales: Cordycipitaceae) and Lecanicillium (=Verticillium ) lecanii (Zimmerman) Viegas. Spray solution s containing 5.4 10 13 conidiospores/ha killed up to 37 % of pear psyllid nymphs in pear orchards in West Virginia (Puterka 1999; Puterka et al 1994). Recently, another study reported that different strains of fungi were pathogenic against the potato psyl lid, Bactericera cockerelli (Hemiptera: Triozidae) (Lacey et al. 2009). Laboratory studies showed that I. fumosorosea (strain Apopka PFR 97) and M. anisopliae (strain F 52) ki lled 95 99% of adults in 2 3 days and 91 99% of nymphs 4 days after application ( Lacey et al 2009). Isaria fumosorosea shows particular promise for use against D. citri in citrus groves in Florida. S trains of I. fumosorosea are highly active against whiteflies and
21 have been sold as a biological pesticide for this purpose in Europe ( Faria and Wr a ight 2001; 2007 ). Also, a strain of I. fumosorosea was found naturally infecting an adult Asian citrus psyllid collected from the underside of foliage on orange trees in Polk County, Florida so this pathogen may be considered native (Meyer et al 2008). In addition, recent studies show that I. fumosorosea does not normally infect natural biological control agents of psyllids and thus would most likely be suitable in citrus IPM programs (Avery et al 2008; 2009).
22 Figure 1 1. Symptoms of HL B in citrus foliage (upper panel) and fruit, and the vector D. citri (lower right). Photograph s courtesy of Tim R. Gottwald and Steve M. Garnsey, USDA, ARS, U.S. Horticultural Research Laboratory 2120 Camden Road, Orlando, FL 32803. Other members of the R utaceae can serve as alternate hosts for HLB.
23 Figure 1 2. Schematic showing how entomopathogenic fungi infect their insect host s through their cuticle s. S ource: Nature Reviews Microbiology 5: 377 383 (May 2007).
24 CHAPTER 2 MATERIALS AND METHOD S All exper iments were conducted at the University of Florida Mid Florida Research and Education Center (MREC) in Apopka The potential of the entomopathogenic fungus Isaria fumosorosea as a n integrated crop management tool for managing D. citri was investigated. In the first phase of the project (Objective 1 ) laboratory experiments were used to determine the pathogenicity and virulence of I. fumosorosea strains applied to D. citri under optimum environmental conditions for the fungus A bioassay system using a me chanized spray tower and detached citrus leaf bioassay was used for this screening procedure. In the second phase (Objective 2) greenhouse tests assess ed the psyllid mortality on orange jessamine p lants that were sprayed with aqueous suspensions of I. fum osorosea An additional aim of the greenhouse study was to investigate the value of incorporating various spray adjuvants along with fungal suspensions Most fungal spores including I. fumosorosea (Kim et al 2011) are lipophilic. Formulation with certa in types of oils can dramatically improve deposition of the entomopathogenic fungi on the insect cuticle as well as its germination and rain fastness ; hence, formulation will significantly improve its effectiveness in the field (Inglis et al 2002). Two hy potheses were tested: (1) strains of I. fumosorosea will have different pathogenicities and virulence to D. citri and (2) the use of oils as formulating ingredients will improve the effectiveness of I. fumosorosea spores applied as a biological pesticide to control D. citri in potted orange jessamine and hybrid Benton citrange ( Citrus sinensis x Poncirus trifoliata ). An insecticidal standard was also included in initial tests for
25 comparison purposes. The insecticidal standard was confirmed to be highly ef fective and was dropped in the last test. Objective 1. Determine the Pathogenicity and V irulence of I. f umosorosea S trains to D. citri Fungal I solates Five treatments (listed below) were screened: One control plus f our commercial strains or formulations of Isaria fumosorosea were assayed to test the pathogenicity and virulence of each against D. citri under op timum conditions for the fungus; treatment 5 was a control All of the commercial strains were originally isolated from whiteflies Bemisia and /or mealybugs, Phenacocus solani Each treatment was a dose that consisted of six leaf dis c s 1. Control: sterile water + Tween 80 (0.025% vol/vol) (Fisher Scientific, Fairlawn, NJ). 2. Isaria fumosorosea : Apopka 97 (PFR 97 20% WDG, Certis, USA, Colombia, MD) a wat er dispersible granule blastospore formulation + Tween 80 (0.025% vol/vol). 3. I. fumosorosea : Apopka 97 (Certis USA) a conidial experimental formulation + Tween 80 (0.025% vol/vol). 4. I. fumosorosea : ARSEF 3581, (USDA ARS, NCAUR, Peoria, IL) a blastospore e xperimental formulation + Tween 80 (0.025% vol/vol). 5. I. fumosorosea : FE 9901, produced as NoFly in Europe, (Natural Industries, Inc., Houston, TX) a blastospore formulation + Tween 80 (0.025% vol/vol).
26 Viability tests were performed on each strain at th e beginning of the study by streaking 0.5 ml of a suspension containing 10 5 spores per ml onto potato dextrose agar (4 replicate Petri dishes) and counting germinated spores that produced a clear germ tube after 19 hours at 25 C. Viability rates were good for ARSEF 3581 ( 99%), Apopka 97 blastospore formulation (79.4%) and FE 9901 (85%), but was low in the conidial formulation of Apopka 97 (24.8%). A n auto load Potter Precision Spray Tower (Burkard Scientific Ltd., Uxbridge, Middx, UK) was used to apply controlled dosages of fungal spores to leaf discs. Fungal suspensions were prepared in sterile water containing 0.025% vol/vol Tween 80 Stock spore suspensions were obtained by concentrating extracts from fungal materials. To achieve the required concent rations 2.5 g of fungal materials were suspended in 20 ml water, vortexed for 1 minute, sonicated for 2 minutes to help dislodge spores from the substrate (model FS20, Vollrath, Sheboygan, WI) and then filtered through 2 layers of cheese cloth to remove larger inert materials. The remaining suspensions (approximately 6 ml) were vortexed a second time and diluted through serial dilutions to approximately10 6 spores/ml in sterile water containing 0.05% vol/vol Tween 80 C ounting was accomplished using an im proved Neubauer hemacytometer (Hausser Scientific, Horsham, PA) and compound microscope at 100 magnification. Based on hemacytometer counts, test solutions containing 10 3 10 4 10 5 10 6 10 7 10 8 and 10 9 spores/ml were created by serial dilutions in ster ile water + 0.025% vol/vol Tween 80 Controls contained sterile water and Tween 80 only. The highest concentration of the 9901 strain was not used because the formulation clogged the spray nozzle.
27 Marsh t because the leaves had a large surface area and a large mid vein to allow psyllid feeding and the leaves were known to be free from pesticide residue. The original certified clean psyllid colony that was supplied by the USDA field station in Ft. Pierce was maintained in an isolated greenhouse on grapefruit. Two cm diameter leaf discs were removed with a sterilized corkborer from 1 week old leaves of young grapefruit trees cv. Marsh grown in an isolated greenhouse. Leaf discs were placed on saturated cot ton wool (to prevent them from drying) in a 9 cm diameter Petri dish and placed at the bottom of the spray tower. A f ive ml sample of each fungal suspension was shaken and loaded at the top of the spray tower s and samples were sprayed at 15 psi starting wi th lowest concentrations to minimize the risk of contamination. Control discs were sprayed first. Each sprayed leaf disc was allowed to surface dry before placing it into a 1 oz plastic cup containing 10 ml of water agar (1.5% w/v, Difco Detroit, MI) and capping it with a tightly fitting lid. The water agar prevented the leaf discs from desiccation throughout the test. Five adult psyllid s, from a colony maintained in a greenhouse in isolation, were aspirated into a clean individual vial then transferred t o one cup. The cups were placed in an incubator set to 26 1 C, 80% relative humidity and 16L: 8D photo period Psyllid mortality and mycosis, hyphal growth and sporulation were recorded daily for 12 consecutive days Statistical Analysis Six replicate leaf discs per fungal concentration were in each bioassay ( hence, 30 psyllids per treatment) and each bioassay was conducted on at least 3 separate occasions for each fungal strain used. Response curves showing the proportions of
28 healthy versus dead and in fected psyllids in relation to dose were subjected to probit regression analysis to determine the lethal concentration to kill 50% of the population. In addition, comparisons of the average survival time (days) of infected and control psyllids using Kaplan Meier survival estimates were used as a measure of virulence. Differences in mean survival times were tested for significance using a log rank test ( SAS 2004 ) with SPSS statistical software Objective 2. Assessment of D. citri Mortality U sing I. fumosorosea with and w ithout E mulsifia ble Oils in Greenhouse T ests. Bioassay P rocedure tree inside a secure cage that served as the mother colony for the bioassays. Meanwhile, clean Murraya plant s were produced from seed in the greenhouse and allowed to grow to the size of 60 cm in height and were then transplanted into 3.8 L pots with sterile potting soil mix containing 55% peat, pine bark, perlite and vermiculite (2B, Fafard, Agawam, MA). Plants were fertilized with slow release citrus fertilizer (12 5 8 Vigoro, The Scotts Company LLC, Marysville, OH) and maintained in greenhouse cages inside of which air temperatures ranged from 16.1 41.4 C (average 25.2 C) and 15 100% r.h. (average 77.7%). Th ese served as host plants for the 2010 greenhouse trial. Two weeks prior to testing, the plants were pruned to encourage new shoot flushes. At the start of the first test conducted in the fall of 2010, 36 orange jessamine plants were placed individually in 60 cm square cages (PVC frame with fitted nylon mesh cover) and each plant was infested with 20 adult psyllids aspirated from the colony. Examination of psyllids showed a sex ratio of 23:77 male: female (N=73). The
29 insects were left for 10 days in the g reenhouse allowing them to mate and oviposit on the plants and for nymphs to emerge. There were six treatments: 1. Control : distilled water and Tween 80 (0.025% vol/vol) 2. Imidacloprid (3 ml product/plant) (Merit 2F Bayer Crop Science, Research Triangle Park NC). 3. Emulsifiable vegetable oil (2.5% vol/vol) (Addit Koppert Biological Systems, AD Berkel and Rodenrijs The Netherlands) + 0.025% vol/vol Tween 80 4. Isaria fumosorosea ( 2.1 g product/L) (PFR 97 blastospore strain) + 0.025% vol/vol Tween 80 5. Isaria fumosorosea ( 2.1 g product /L) (PFR 97 blastospore strain) + 2.5% vol/vol Addit + 0.025% vol/vol Tween 80 6. Highly refined paraffinic oil (2% vol/vol) (SuffOil X BioWorks, Victor, NY) + 0.025% vol/vol Tween 80 The PFR 97 was weighed (2.1 g/L) and mixe d in approximately 100 ml distilled water for at least 30 minutes prior to use. The final rate applied was equivalent to 8 .28 mls product/ 3.8 L (upper label rate). A standard viability test indicated that spores suspended in water germinated at a rate of 8 2% when cultured on PDA media for 20 hours at 25C. However, the impact of emulsifiable oils on spore germination was not specifically assessed in this study. Also, since product s were applied at label rates (weight), actual spore concentration per ml was not counted in greenhouse tests (see discussion). Emulsifiable oil treatments were applied with and without Isaria Addit was used since it is recommended for use with another entomopathogenic fungus to have good compatibility. SuffOil X was applied within label rate (labeled rate = 1 2% vol/vol). SuffOil X is labeled for citrus in the U.S. for control of whiteflies, scales, mites and mold. Imidacloprid (Merit 2F ) was used as an insecticide standa rd. It was applied as a soil
30 drench (3 ml product per plant diluted in 75 ml water) using a watering can. Distilled water served as the untreated control. Tween 80 was added to all foliar treatments as a surfactant. seconds using a 3.7 Liter hand held pressurized sprayer (Flow master 1401WMX, Root Lowell Manufacturing Co., Lowell, MI). The sprayer output was calibrated to 3.88 ml/s. Each plant received 38 ml of inoculate (not all went on the plant). All foliar trea tments were reapplied after 7 days. There were 6 replicate plants in each treatment group arranged in a completely randomized design inside a single greenhouse bay. The treatments were applied late afternoon/early evening to take advantage of higher relat ive humidity at that time of day. An overhead misting system was operated for 20 seconds after all treatments were applied and was shown, using dataloggers inside cages (HOBO U10, Onset Computer Corp., Cape Cod, MA), to maintain > 95% r.h. in the greenhous e for 8 hours. The mister was used to elevate the ambient air humidity and to encourage fungal spores to germinate but it did not significantly wet the leaves of the plants inside the cages. In the first experiment, the psyllid populations on the Murraya plants were counted initially as a baseline prior to any spray applications and then every seventh day for four consecutive weeks. The total number of psyllids (adults and nymphs) w as counted from a minimum of three shoot terminals per plant. In the fourt h week, the plants were destructively sampled and a final count was taken. P syllids showing signs of fungal sporulation were collected, transferred to PDA medium under sterile conditions and monitored for fungal outgrowths. After 2 weeks incubation, the fungal colony was
31 postulates. Temperatures throughout the 2010 greenhouse tests ranged from 16.1 41.5C (mean 25.2C) with a relative humidity between 15 100% r.h (mea n 77.7 % ). The greenhouse experiment was repeated in summer and fall of 2011, however, Benton citrange, [ Citrus sinensis (L.) Osb. x Poncirus trifoliate (L.) Raf] plants were used instead of Marsh grapefruit. This change in host plants was due to an inabi lity to acquire Marsh grapefruit plants that had not been treated with imidacloprid. The summer treatments were: 1. Control : distilled water + Tween 80 (0.025% v ol /v ol ) 2. Merit 2F (3 ml product/plant) 3. Addit (2.5% v ol /v ol ) + Tween 80 (0.025% v ol /v ol ) 4. PFR 97 (2.1 g product/L) + Tween 80 (0.025% v ol /v ol ) 5. PFR 97 (2.1 g product/L) + Addit (2.5% v ol /v ol ) + Tween 80 (0.025% v ol /v ol ) 6. SuffOil X (2% v ol /v ol ) + Tween 80 (0.025% v ol /v ol ) As additional modifications in the fall 2011 greenhouse test there were 5 r eplicate plants per treatment a nd slightly different oil combinations were tested Imidacloprid (Merit 2F ) was not included since its high efficacy was clearly shown in the 2010 and summer 2011 greenhouse tests. Orocit (Oro Agri Inc., Dallas, TX) an adj uvant containing alcohol ethoxylate, was evaluated to determine if it would improve fungal pathogenicity of Isaria Orocit is currently labeled for citrus as an adjuvant to improve efficacy of miticides, insecticides, fungicides and herbicides. SuffOil X was applied at 1% v/v to determine if it was also effective at the lower label rate. The fall 2011 treatments were: 1. Control : distilled water + Tween 80 (0.025% vol/vol)
32 2. Orocit (0.25% vol/vol) + Tween 80 (0.025% vol/vol) 3. SuffOil X (1% vol/vol) + Twee n 80 (0.025% vol/vol) 4. PFR 97 (2.1 g product/L) + Tween 80 (0.025% vol/vol) 5. PFR 97 (2.1 g product/L) + Orocit (0.25% vol/vol) + Tween 80 (0.025% vol/vol) 6. PFR 97 (2.1 g product/L) + SuffOil X (1% vol/vol) + Tween 80 (0.025% vol/vol) The germination rat e of the fungal spore used in these tests w as determined to be 66 77% after 20 hours at 26C on PDA plates. This germination was assessed prior to mixing with the various oil additives. Temperatures throughout the summer 2011 greenhouse tests ranged from 22.2 39.7C (mean 28.8C) with a relative humidity between 42 100% (mean 83.0%). Temperatures throughout the fall 2011 greenhouse tests were slightly cooler and less humid, and ranged from 18.5 40.9C (mean 25.0C) with a relative humidity between 15 100% (mean 69.1%). Statistical Analysis The numbers of infested terminals and live psyllid (adults and nymphs) were compared between fungus and control and chemical treatments through analysis of variance (ANOVA) in a randomized design following log ( n +1) tran sformation. Where appropriate, means were further compared with Fishers protected LSD tests at P < 0.05.
33 CHAPTER 3 RESULTS Stages of D evelopment of I. fumosorosea In V itro and in D. citri Blastospores from I. fumosorosea rapidly developed germ tubes on c u lture media (Fig. 3 1A, B) and subsequently sporulated (Fig. 3 1C and D). Symptoms of inoculated psyllids included twitching of legs and antennae 1 2 days before death. Immediately following death, infected psyllids had fungal hyphae emerging from the tars i and intersegmental regions of the legs (Fig. 3 1E ). Within 24 48 hours post mortem, significant mycelial growth developed on the dead insect (Fig. 3 1F), f ollowed by development of phailides (Fig. 3 1G) and conidiogenesis (Fig. 3 1H). Survival and M ycos is of D. citri in L aboratory B ioassays Adult psyllids died at different rates over 12 days (Fig 3 2). By day 12 some of the control psyllids (approximately 30 45%) ha d also died, probably as a result of the bioassay conditions and natural age related mort ality. However, the bioassay approach was still successful since when compared with controls survival was significantly reduced following exposure to all strains of I. fumosorosea with a clear concentration response in all cases. Overall, 100% of psyllid s died within 12 days at the higher concentrations, while generally > 80% of psyllids died at the lower concentrations. Average survival times calculated from the data showed differences between the strains, notably the fastest mortality was observed for t he AR SEF 3581, followed by FE 9901 and the Apopka 97 blastospore formulation (Table 3 1). Probit analysis conducted on the fungus concentration response data showed that the lethal dose required to kill 50% of the psyllids after 7 days was in the range of 1 2 10 5 blastospores/ml for ARSEF 3581 and FE 9901, and significantly higher 2 10 6 blastospores/ml for
34 Apopka 97 (Table 3 2). A higher value again (based on non overlapping confidence intervals) was obtained for the conidial formulation of Apopka 97. A 7 day exposure period was selected since control mortality was still relatively low at this time (< 20%) hence results would reflect the effect of the fungal exposure. The proportion of psyllids expressing symptomatic response to fungus was also differe nt according to the fungus strain and concentration applied to the leaf discs (Fig 3 3). In general, up to 100% of psyllids sporulated at concentration s of 10 8 or higher but declined at lower concentrations. This decline was most apparent in the conidial formulation of Apopka 97 strain where < 30% psyllids became symptomatic at concentration s of 10 5 conidia/ml or less. Evaluation of F oliar S prays of I. fumosorosea and E mulsifiable O ils a gainst D. citri I nfestations on C itrus u nder G reenhouse C onditions In the fall 2010 test, the induced flush ensured oviposition and large numbers of D. citri nymphs were present when treatments were first applied, and 2 nd generation (F 1 ) adults were present by week 2 (Table 3 3). All treatments significantly reduced these F 1 ACP adults compared to the control treatment by weeks 2 and 3. Merit was the most effective treatment followed by SuffOil X and PFR 97, Addit and PFR 97+Addit (Table 3 3). The combination of PFR 97 and the emulsifiable oil did not increase ACP mortality compared with either agent alone. Only Merit and SuffOil X significantly reduced the number of infested terminals (on enclosed plants) from week 2 4. In the destructive count, Merit 2F was the most effective with 99.9% reduction with respect to the untrea ted control (UTC) followed by SuffOil X (85.6% reduction), Addit (56% reduction), PFR 97 (52.2% reduction), PFR 97+Addit (49.8% reduction) (Table 3 postulate test, slightly increased mortality was observed from adult ACP exposed to
35 funga l suspensions compared with controls after 7 days ( i.e. 26.7% 6.7 versus 9.6% 5.7), however this difference was not significant (F1,10 = 2.35, P = 0.16). An average of 20% of psyllids that died following exposure to fungal suspensions and incubated in the laboratory produced outgrowth consistent with I. fumosorosea symptoms confirming that I. fumosorosea was present, although the mortality rate was lower than expected. In the 2011 tests, contamination by Cladosporium sp. was observed in all treatments (Fig. 3 4) In the summer test, while all treatments reduced the number of ACP nymphs by week 1 with Merit being the most effective, relatively few F 1 adults were subsequently observed in control cages (Table 3 4). Observations showed widespread growth of Cladosporium among all treatments, which compromised the ability to determine treatment effects. A similar observation was found in the fall 2011 test (Table 3 5), where treatments significantly reduced live nymph counts on week 1 and 2, but numbers of F 1 adults remained low. Overall, treatments containing oils alone or in combination with PRF were most effective in the fall 2011 test.
36 Figure 3 1. Photographs showing stages of development of I. fumosorosea in D. citri (a) Non germinated blastospores 1 00 magnification, (b) blastospore germination on PDA media 100 magnification, (c) and (d) colony forming units on PDA, (e) initial and (f) late mycelial growth protruding from dead insect 40 magnification, (g) chains of conidiophores developed on phail ides, note whorled branching of hyphae with conidia 400 magnification, (h) sporulating insect cadavers 40 magnification. Photo credits (F and G) Pasco Avery
37 Figure 3 2. Proportional survival of adult D. citri following exposure to citrus leaves trea ted with different formulations of Isaria fumosorosea at concentrations between 10 3 and 10 9 spores per ml. Da ta are mean SEM of 3 tests (40 psyllids per test).
38 Figure 3 3. Proportion mycosis (sporulation) of adult D. citri following exposure to Marsh g rapefruit leaves treated with different formulations of Isaria fumosorosea at concentrations between 10 3 and 10 9 spores per ml. Da ta are mean SEM of 3 tests (40 psyllids per test)
39 Figure 3 4. Cladosporium sp. contamination observed on Benton citrang e in greenhouse cages.
40 Table 3 1. Average survival times (days) of adult D. citri exposed to 4 strains/formulations of Isaria fumosorosea at concentrations between 10 3 and 10 9 spores per ml. Strain Concentration (spores/ml) 0 10 3 10 4 10 5 10 6 10 7 10 8 10 9 ARSEF 3581 10.1b 9.1a 8.2a 7.1b 6.0c 4.5c 3.5d 3.1c Apopka 97 blastospore 10.2b 9.4a 8.3a 9.1a 7.5b 6.1b 4.4b 3.9b FE 9901 10.9a 10.2a 8.4a 7.3b 6.1c 4.6c 4.1c NA Apopka 97 conidial 10.3ab 10.1a 10.0a 9.4a 8.9a 6.7a 5.1a 4.7a M ean s based on 3 tests ( 30 psyllids per test). Estimates determined by Kaplan Meier Survival analysis, with data for survivors censored at day 12. Letters in columns indicate significant differences (P < 0.05) between strains according to a log rank (Mantel Cox) test.
41 Table 3 2. Estimates of the median lethal concentration (spores/ml) of three entomopathogenic fungi applied against adult D. citri on Marsh grapefruit leaf disc assays at 7 days post inoculation. Strain LC 50 95% C L Slope SEM 2 ARSEF 3581 1.37 10 5 5.1 10 4 3.2 10 5 0.56 0.15 0.21 Apopka 97 2.03 10 6 7.4 10 5 4.8 10 6 0.54 0.12 1.18 FE 9901 1.36 10 5 5.4 10 4 3.0 10 5 0.32 0.08 1.25 Apopka 97 conidial 1.47 10 7 5.6 10 6 3.4 10 7 0.32 0.06 2.35 Data based on 3 tests ( 30 psyllids per test); all probit estimates were adjusted for control mortality.
42 Table 3 3. Number of infested terminals and ACP recorded on orange j essamine plants following treatment applications in the fall 2010 test In fested terminals/plant ACP adults/terminal shoot ACP nymphs/terminal shoot Treatment Rate Pre treat Week1 Week2 Week3 Pre treat Week1 Week2 Week3 Pre treat Week1 Week2 Week3 Control 7.5a 10.5a 11.8a 16.8a 1.0a 0.9a 16.1a 16.2a 24.1a 30.3a 15.0a 1. 8a Merit 2F 3ml/plant 10.7a 6.2c 0.5c 0.2c 0.6bc 0.0a 0.0c 0.1d 17.2a 4.6c 0.2c 0.0b Addit 0.25% v/v 12.0a 10.3a 10.7ab 14.0a 0.4c 0.7a 3.4b 5.5b 27.8a 16.2b 8.9ab 1.3a PFR 97 0.28 oz/gal 10.2a 10.3a 10.2ab 15.0a 0.7abc 0.2a 5.4b 5.0b 26.7a 19.3a b 7.9ab 1.9a PFR 97 + Addit 0.28 oz/gal + 0.25% v/v 9.0a 9.3ab 8.5ab 11.5ab 0.8ab 0.2a 3.4b 4.7b 24.8a 21.4ab 7.8b 1.1a SuffOil X 2% v/v 10.7a 6.8bc 7.0b 7.2b 0.6bc 0.2a 2.6bc 2.3c 30.6a 8.6c 4.4b 0.7ab Letters in columns indicate differences ( P > 0 [log10 (x+1)]; non transformed means shown.
43 Table 3 4. Number of infested terminals and ACP recorded on Benton citrange plants following different treatments (summer 2011 test) Infe sted terminals/plant ACP adults/terminal shoot ACP nymphs/terminal shoot Treatment Rate Pre treat Week1 Week2 Pre treat Week1 Week2 Pre treat Week1 Week2 C ontrol 8.3a 9.5a 7.7ab 0.6a 0.7a 2.4a 36.4a 12.7a 2.8a Merit 2F 3ml/plant 9.7a 5.6a 1.0c 0.5a 0.0a 0.0d 43.1a 1.1c 0.0c Addit 0.25% v ol /v ol 8.5a 8.2a 9.7a 0.4a 0.7a 0.8bcd 32.1a 4.8b 2.1ab PFR 97 0.28 oz/gal 9.0a 7.2a 7.7ab 0.3a 0.8a 1.1abc 38.0a 4.2b 0.6bc PFR 97 +Addit 0.28 oz/gal + 0.25% v ol /v ol 7.3a 5.2a 6.2b 0.5a 0.7a 1.4ab 26.2a 4.6b 1.6ab SuffOil X 2% v ol /v ol 8.3a 6.9a 6.8ab 0.3a 0.7a 0.3cd 32.9a 4.8b 0.3bc [log10 (x+1)]; non transformed means shown
44 Table 3 5. Number of infested terminals and ACP recorded on Benton citrange plants following different treatments (fall 2011 test) Treatment Rate Infested t erm inals/plant Ad ult Nymph Pre treat Week1 Week2 Week3 Pre treat Week1 Week2 Week3 Pre treat Week1 Week2 Week3 Control 7.8a 11.2a 10.4a 9.4a 1.0a 0.9ab 1.2a 1.5ab 14.1a 34.1a 20.7a 6.5a Orocit 0.25% v ol /v ol 7.6a 6.2bc 3.8b 4.0c 0.5a 0.2bc 0.0b 0.2c 28.7a 17.5b 3.2c 1.7b SuffOil X 1% v ol /v ol 7.0a 5.4c 4.4b 4.8bc 0.5a 0.2bc 0.1b 0.3c 18.3a 15.7 b 2.1c 0.9bc PFR 97 0.28 oz/gal 8.4a 9.0ab 7.6a 6.6ab 0.7a 1.9a 1.0a 1.7a 20.1a 19.3b 9.4b 1.7b PFR 97 + Orocit 0.28 oz/gal +0.25%v ol /v ol 7.6a 7.6abc 4.6b 4.6bc 0.8a 0.0c 0.1b 0.7bc 21.3a 18.9b 3.5c 1.7b PFR 97 + SuffOil X 0.28 oz/gal + 1% v ol /v ol 7.8 a 6.2bc 3.4b 4.2bc 0.5a 0.0c 0.2b 0.3c 16.7a 8.9c 1.2c 0.3c [log10 (x+1)]; non transformed means shown
45 Table 3 6. Number of ACP on or ange jessamine plants as determined by a final destructive count (fall 2010 test) Treatment Rate ACP adults/plant ACP nymphs/plant Total/plant Control 292.7a 11.3a 304.0a Merit 2F 3ml/plant 0.2d 0.0c 0.2d Addit 0.25% vol/vol 130.3b 3.5ab 133.8 b PFR 97 28 oz/100 gal 137.0b 8.3a 145.3b PFR 97 + Addit 28 oz/100 gal + 0.25% vol/vol 144.0b 8.5a 152.5b SuffOil X 2% vol/vol 41.5c 2.2bc 43.7c transformed due to une qual variances [log10 (x+1)]; non transformed means shown.
46 CHAPTER 4 DISCUSSION Renewed interest in the use of entomopathogenic fungi to manage insect pests and the discovery of a strain of I fumosorosea (= Paecilomyces fumosoroseus ) naturally infecting D. citri in a Florida citrus grove led to this study to further assess the potential of I fumosorosea for use to manage D. citri Presently three strains of I fumosorosea are available for research as blastospore formulations in the USA Apopka 97 which is formulated as a commercial material PFR 97 WDG (Certis USA) for control of soft bodied insects including aphids, mites and whiteflies ; FE 9901 which is supplied by Natural Industries Inc. as NoFly bioinsecticide with registration status pending ; and ARS EF 3581 supplied by USDA ARS, NCAUR, Peoria Illinois in diatomaceous earth formulation (Jackson et al. 1997). A simple method to quantify the pathogenicity and virulence of I fumosorosea strains under optimum conditions was used In developing this meth od, we found that the use of disposable opaque cups successfully maintained high, humid conditions necessary for the fungal isolate development and sporulation. Additionally, the cups supported a detached leaf diet sufficient enough to keep five adult ps yllids alive for a period of up to twelve days while maintaining leaf turgor Control mortality remained low (< 20%) after 7 days, although it increased to higher than preferred levels (30 45%) after 12 days. The sealed cups minimized contamination and sta ndardized light and temperature conditions throughout the bioassay. L aboratory conditions are artificial, thus, it was important to quantify the impact of the fungus under more realistic conditions. We noted that all of the fungal strains reduced the surv ival of adult psyllid by up to 100% We selec ted a commercial strain (Apopka 97) for greenhouse tests. The Apopka 97 blastospore strain was originally
47 isolated from mealybugs at MREC (Osborne and Landa 1992) and is now commercially marketed as PFR 97 WDG by Certis, USA (Menn 2003) registered for control of soft bodied insects including aphids, mites and whiteflies on food crops including citrus. Since v arious petroleum and organic derived oils are used in citrus groves in Florida for control of soft bodied insects and disease management (Rogers et al. 2011 ) we also chose to include them in this research The oils used in this study are used by growers in Europe and United States. In the first greenhouse test, we observed that PFR 97 reduced psyllid populat ions by approximately 50% over 3 weeks while the oil treatments ranged from 56 8 5% mortality Additionally in the 2010 test the petroleum based oil (Suffoil X ) was significantly more effective at reducing psyllid numbers when compared with the vegetable based oil (Addit ). The reason for these differen t impacts are unknown, but may relate to the higher rates applied for the former, as well as the superior coverage of the high ly refined paraffinic material. It was hypothesized that formulating blastospores with emulsifiable oils might improve control compared with the fungus alone. Previous studies have highlighted several benefits of formulating entomopathogenic fungi with oils ; these benefits enhanced not only deposition on the insect cuticle, but also ge rmination rate and rain fastness (Inglis et al. 2002). The effectiveness of Apopka 97 in our tests was not enhanced by adding emulsifiable oils and did not increase psyllid mortality compared with oils used alone. The reasons for the lack of synergy are un known. However one possibility is that the oils negatively affected germination or penetration of blastospores. Unfortunately this possibility was not specifically tested in this study. In 2011, Kim et al. assayed carriers to enhance persistence of Isaria fumosorosea They found that a corn
48 oil carrier was superior in maintaining germination rates of I. fumosorosea when compared with other oils such as soybean oil, cotton seed oil, paraffin oil, and methyl oleate. The variability in different oil traits nee ds to be studied further with combined fungus and oil treatments. Entomopathogenic fungi effectiveness may also be inconsistent due to abiotic factors such as temperature and humidity and biotic factors like interactions of antagonistic microorganisms (F erron 1978; Villani et al 1992). It can be speculated that increasing the period of hydration of blastospores for several hours prior to application may improve the germination success on the target insect. In follow up tests conducted in 2011, there were several difficulties in assessing the fungus under greenhouse conditions, mainly because of high mortality in all treatments and the widespread contamination with Cladosporium sp. that was observed several weeks into the study. Previous studies have shown that adult and nymphal mortality rates are higher where a minimum daily relative humidity is high ( tienne et al. 2001 ; Hall et al 2008 ; Meyer et al 2008). It may be that high mortality in controls was due to the spreading or natural occurr ence of I. fu mosorosea in the greenhouse which was encouraged by the very high humidity (i.e. in summer 2011 tests r.h. was > 90% in > 50 % of hourly recordings). However, this hypothesis could not be confirmed since Cladosporium sp. grew on almost all insect cadavers and honeydew deposits. Thi s growth was thought to be saprophytic since Cladosporium sp.did not kill psyllids at 10 7 conidia/ml in the laboratory. T here is a previous report that Cladosporium sp. nr. oxysporum Berk. Infected D. citri in Runion Island (Au bert 1987). Avery et al (2004) isolated Cladosporium sp. on whitefly nymphs infected with Trinidadian strains of I. fumosorosea
49 PFR is a known entomopathogen of ACP (Hoy et al. 2010 ; Hunter et al. 2011) and other insects including whiteflies, aphids, thr ips and spider mites. Avery et al. in 2011 studied the effects of two PFR 97 formulations (blastospores and conidia) on the ACP They monitored feeding rates and adult mortality through a laboratory bioassay and discovered that after 7 days post exposure, total mortality occurred from both isolates. The blastospore formulation caused a significantly higher mortality than conidia within the first 3 days. They also documented that infected adult psyllid produced less honeydew than healthy psyllids suggestin g that a reduction in feeding activity could potentially reduce the spread of huanglongbing. Avery et al (2011) speculated that the use of Isaria which is specific to insects, is valuable because it reduces feeding by healthy psyllids, prevents infected psyllid s from spreading the disease and allows infecte d psyllids to serve as inoculums and spread the fungus throughout the citrus groves, thus killing even more psyllids He developed that increased horizont al transfer of PFR and suggested that its adaptation for use in the grove could reduce HLB dissemination and potentially reduce costs to growers (Avery et al. 2009). S everal additional entomopathogenic fungi are known to infect D. citri including Lecanic illium lecanii (= C ephalosphorium lecanii ) (Rivero Aragon and Grillo Ravelo 2000; Xie et al. 1988), Beauveria bassiana (Bals.) Vuill. (Rivero Aragon and Grillo Ravelo 2000), Capnodium citri Berk and Desm (Aubert 1987) and Hirsutella citriformis Speare ( ti enne et al. 2001; Meyer et al. 2007 ; Rivero Aragon and Grillo Ravelo 2000; Subandiyah et al. 2000 ). A previous report dating back to 1987 indicates that
50 Cladosporium sp. nr oxysporum Berk. Infect D. citri in Reunion Island (Aubert 1987). Entomopathogens are considered importan t factors of psyllid mortality. Potato psyllid, Bactericera cockerelli is a serious pest of potato and other solanaceous vegetables also transmit a bacterium, Candidatus Liberibacter solanacearum that et al. (2009) observed under ideal laboratory conditions that two isolates of Metarhizium anisopliae and two isolates of Isaria fumosorosea caused > 95% mortality of adult potato psyllid. A Beauveria bassiana isolate provided 53% mortality after 2 3 days. The mortality rate of Isaria isolates in the laboratory was similar to our findings. In further field tests in Texas, treatment with M. anisopliae (F 52) PFR 97 and abamectin (Agri Mek) significantly decreased plant damage and zebra chi p symptoms in a potato field ( Lacey et al. 2011). Casique Valdes et al. (2011) confirmed in their laboratory studie s that Hirsutella cf. citriformis fungal isolates provide a viable component for an integrated pest management (IPM) strategy for control of D. citri They successfully isolated H. citriformis from ACP and found it to be pathogenic to adults of D. citri the potato psyllid, B. cockerel and Nilaparvata lugens (brown leafhopper). T amarixia radiata (Waterston) is a classical biological ly introduce d ectoparasitoid of ACP from India ( tienne et al. 2001 ; Hoy and Nguyen 2001 ; Hoy et al. 2006 ; Waterston 1922 ). It is one of many predators and parasitoids that keep psyllid nymphs in check ( Chien et al 1991 ; tienne et al. 2001 ; Halbert and Manjunath 20 04 ; Hoy and Nguyen 2001 ; Len and S tamou 2010 ; Michaud 2004 ; Pluke et al. 2008; Waterston 1922) However the amount of psyllid control provided by introduced parasitoids has been insufficient to slow disease spread.
51 Michaud ( Michaud and Olsen 2004) found that other predators are equally efficient in controlling psyllids. Through the use of field cages, he found that, although T. radiata contributed to mortality of the psyllids, coccinellid beetles, such as Harmonia axyridis Pallas, were more important bio logical control agents in high density D. citri populations in central Florida. In an IPM program, a priority should be placed on natural mortality of the ACP wherever possible Many pesticides have been shown to be highly toxic to psyllid predators and p arasitoids (Michaud and Grant 2003). Toxicology studies from Hall and Nguyen (2010) indicated that carbaryl, chlorpyrifos and fenpropathrin were lethal to adult T radiata as late as 3 days after application. Systemic insecticides such as imidacloprid are an important part of psyllid control (Rogers et al. 2010 ; Boina et al 2009 ). Imidacloprid (Merit) provides persistent systemic activity against citrus psyllids. Used as a drench or foliar spray, it functions as a broad spectrum insecticide that is active on various Hom optera and leaf beetles (Rogers et al. 2010). Unfortunately, imidacloprid also causes mortality on beneficial insects, including T. radiata through chemical residual effects (Cocco and Hoy 2008). Broad spectrum pesticides have a place in an IPM program; however, current recommendations limit applications to late spring and early fall when psyllid populations are actively reproducing on new flush (Rogers et al 2010). The persistence of Paecilomyces fumosoroseus is significantly affected by relative humidi ty of the drying air. Desiccation and shelf life of blastospore preparations are improved when the relative humidity of the drying air exceeds 50% (Jackson and Payne 2007). High temperatures during storage results in a reduction of shelf life of Isaria. In
52 order for living biological control agents to be useful, they must be stable, economical and provide viable fungal isolates in field conditions.
53 CHAPTER 5 CONCLUSIONS All three I saria isolates were pathogenic to ACP in the laboratory. Apopka 97 provide d 50% or higher mortality on mixed age psyllid populations under greenhouse conditions Foliar applications of oils (SuffOil X Addit and Orocit ) were effective in reducing psyllid populations by a range of 56 85 % over three weeks. It did not appear as if the oils acted synergistically with the Isaria although further research is need ed in this area. Imidacloprid applied as a drench killed 100% of psyllid within 3 weeks in greenhouse tests. There are several factors that can determine how well Isaria w ould work in a commercial grove setting. Important factors will include ambient air temperatures and relative humidity within the citrus grove, air circulation within the canopy, accurate spray equipment and thorough coverage of the foliage during spray a pplications. Continued research is needed on effective, non toxic environmentally friendly insecticides that can help with the fight against the ACP. These tools are vital in an integrated crop management program against HLB
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64 BIOGRAPHICAL SKETCH Kare n Palanuk Stauderman was born in Springfield, Oregon and graduated from Thurston Senior High school i n 1979. She earned a B. S. in plant pathology and a B. S. in h orticulture from Oregon State University. She attend ed graduate school at the University of Nevada Reno and Oregon State University majoring in plant s cience. In 1988, Karen relocated to Florida where she began work with the University of Florida as a biological s cientist at the Central Florida Research & Education Center in Apopka, FL. Karen work ed in the Entomology Department led by Dr. Lance Osborne and assisted in the initial culturing of Isaria After a year, she was promoted to a biological s cientist II in the Plant Pathology Department and was re lo cated t o the UF Sanford Research Center (1989) where she remained for 10 years managing laboratory investigations in cut foliage, carrots and the cabbage leaf curl virus Karen left UF in 2000 to expand a family farm along with her husband Harry and daughter Lynn ( then age 5 ). Their venture, Oak Haven Farms of Mount Dora, LLC (established 1996) and Oak Haven Winery is an agri tourism farm offering U pick strawberries, rest aurant, vineyard and w inery. Upon completion of her M. S. program, Karen will continue her current position with UF as a Horticulture Extension agent with Volusia County in Deland, FL where sh e has been employed since 200 7