The overall goal of this 3-year research project is to efficiently deliver antimicrobial molecules into citrus phloem against HLB bacteria. This quarter’s (From January 2015 to April 2015) research was focused on: 1) continuing to evaluate the effect of antimicrobial compounds and their combinations at different concentration against HLB bacteria using nano formulation by bark-application; 2) investigating agronomic performance and HLB bacterial titer of HLB-affected citrus trees treated with nano-formulations by bark painting and gravity bag infusion in the field; 3) comparing efficiency of different emulsions on capturing insects. In this quarter, thirty-two treatments from 14 antimicrobial compounds in combination and alone at various concentrations loaded in oil in water nanoemulsion, continued to be evaluated in the greenhouse. The results indicated that, 6 month after initial treatment, Amp and Pen were the most effective chemical compounds against the Las bacterium. Las bacterium titer was significantly reduced by Act, Act+Va, and Va. No phytotoxicity appeared in citrus trees treated with Act and Act+Va at lower concentrations. Other antimicrobial compounds and combinations (i,e, Pround, Al(OH)3, SDX) were not effective against the Las bacterium. In field trials, Pen and SDX were delivered into citrus phloem by bag gravity infusion and bark-painting. Using bag gravity infusion, Pen can be effectively delivered into citrus phloem against Las bacterium, and Las bacterium titer was kept low (Ct=33.0-38.5) for 12 month. But in HLB-affected citrus trees treated with Pen by bark-painting, Las bacterium was not significantly reduced. In addition, SDX was not effective against the Las bacterium using either application method. Furthermore, agronomic performance (i,e, fruit size, fruit weight, and Brix) of the citrus trees also was investigated in this quarter. The results showed that fruit size and fruit weight under Pen and SDX treatment, were much greater than those in the control. Brix was not significantly different among treatments. In our previous study, water in oil nanoemulsion (Cre-Nano) has been shown to be an ideal delivery system for enhancing effective compounds to penetrate through citrus cuticles. Effects of three nanoemulsion formulations (Cre-Nano, NanoGreen (business product) and Carv-nano) on capturing insects was also evaluated in this quarter. These three nanoemulsion formulations were applied to citrus trees by foliar spray outdoors. Six days after initial treatment, the citrus tree treated with Cre-Nano captured 10 insects, significantly more than those treated with Nanogreen and Carv-nano. In the next quarter, we will complete this project by submitting the final report.
To improve the RNAi strategy for psyllids control, we used artificial micro RNAs (amiRNAs) to target psyllid mRNAs for better specificity and to lower the potential for off-target effects. Previously, we finished making the clones expressing amiRNAs targeting V-ATPase mRNA of Bactericera cockerelli in two viral vectors and a non-viral vector. We used agroinfection-compatible Tobacco mosaic virus (TMV) and Tomato mottle virus (ToMoV-A, TAV) expression vectors, and a virus-free 35S promoter driven binary vector and delivered amiRNAs through transient expression in plants and through artificial diets. To evaluate the quantity and quality of the amiRNAs expressed in plants (Nicotiana benthamiana), we used Hi-Seq small RNA deep sequencing with 3 replicates. The results showed that the TAV vector and the non-viral vector expressed amiRNAs with greater specificity compared to the TMV vector, and the TAV vector expressed the highest amount of amiRNAs in N. benthamiana plants. The 3 replicated trials showed that the expression efficiency is 100% repeatable and reliable. We also confirmed the expression levels of amiRNAs with Northern blot analysis, and the results were in agreement with those from the deep sequencing analyses. We currently are evaluating the functionality and the efficiency of the amiRNAs targeting to the ATPase mRNAs in protoplasts, to confirm the amiRNAs we express are functional and able to target to the target mRNAs. We are also doing feeding tests with different approaches, plant and artificial diet feeding tests. The target mRNA of the psyllids from the feeding tests was evaluated by quantitative PCR (qPCR). Preliminary data show that the levels of target mRNAs from psyllids fed on the amiRNAs were not stable compared to the control psyllids fed on healthy leaves, possibly due to the various quantities of amiRNAs acquired by each psyllid from leaf tissues. We are working on finishing feeding tests for all different amiRNA expression clones in different diets and to evaluate the target mRNA levels by RT-qPCR. We are also developing transgenic plants expressing amiRNAs for further biological analysis.
We have been investigating the application of synthetic sex pheromones to disrupt mating of the citrus leafminer (CLM) as a possible effective and environmentally friendly alternative to pesticide applications. We have conducted large-scale field experiments combined with data modeling to optimize the use of this technology for the purpose of practical application. Optimizing mating disruption through examination of multiple interrelated variables was a method used for possible wider adoption of this technique in Florida citrus, especially since this particular pheromone active ingredient is quite expensive to produce. Simulations and field experiments were designed to produce response surfaces to investigate data. We varied the distribution and number of pheromone dispensers in plots. Moth catch data in traps were used to understand the efficacy of our treatments. We used commercially available dispensers for mating disruption of CLM obtained from ISCA Tech. For modeling, Monte Carlo simulations were used for a spatially explicit agent-based model. These resulted in nonlinear disruption profiles with increasing point source density. Field trials conducted in citrus infested by CLM varied the amount of pheromone applied at each point source and point source density using attractive and non-attractive disruption blends. Trap catch disruption in the field resulted in nonlinear disruption profiles similar to those observed with simulations. Response surfaces showed an interaction between the amount of pheromone applied and the number of point sources for the attractive blend, but not for the non-attractive blend. Disruption surfaces were combined with cost curves to optimize trap catch disruption under real world cost constraints. Our bottom line results were as follows. Our data indicate that the more complex and more expensive 3:1 blend of two pheromone components was not more economically effective than the less expensive single component blend. Also, our data suggest that both formulations actually cause disruption by the same noncompetitive mechanisms. Therefore, we have definitively shown that the less expensive single component dispenser is the most effective for practical adoption for use to manage CLM in Florida using a combination of filed trials and data modeling.
Densities of an herbivorous pest may be impacted by landscape and orchard architecture. We have been conducting two experiments where the densities of the Asian citrus psyllid (ACP) were compared depending on: (1) the presence or absence of a windbreak and (2) if the groves consisted of a solid set of new plantings or groves with a mixture of mature and reset-replacement trees. (1) Psyllid abundance was measured on the edges of five groves. We have finished analyzing these data in the previous quarter. The factor investigated was the presence or absence of a windbreak. For the five groves, we observed significantly fewer psyllids on the edges of groves with windbreaks as compared to those without windbreaks. We found no significant difference in the number of natural enemies between the edges with or without windbreaks, suggesting that windbreaks do not affect densities of psyllid natural enemies. (2) During two consecutive years, we compared the densities of psyllids on young trees less than 3 years of age in a solid set re-planting versus on resets (trees planted in replacement of dead or huanglongbing-infected trees) present randomly within mature groves. This was conducted in four groves and among three citrus varieties. These data have now been analyzed. More psyllids were found in the solid set new plantings as compared with on the resets within mature groves. Overall our data suggest that establishment and conservation of windbreaks might be beneficial to protect groves from ACP. The data also suggest that ACP populations increase more within uniform landscapes of seedling trees as compared with mature groves with randomly interspersed young seedlings. We consistently found that young trees replanted as solid sets contain higher populations of ACP than seedling trees replanted as resets within otherwise mature orchards. These data may indicate that replacing individual infected trees with uninfected seedlings in a mature grove structure may be a more effective method to prevent infestation by ACP (and associated HLB re-infection) than replacing entire blocks of orchards. Changes in microclimate may also reduce the population of ACP on young citrus resets compared with young plantings in the solid sets configuration. We found that canopy temperature was greater, on average, in young trees present within solid sets as compared with resets re-planted within mature orchards. This increase in temperature was particularly evident in the morning (up to 4′ C) and is probably correlated to light exposure at this time of the day in Florida. Sun exposure is likely to have a strong impact on ACP colonization of plants and dramatically differs among trees depending of their size and the depth of the canopy. Presence of mature trees surrounding young trees likely reduces wind and sun exposure on young citrus trees analogously to a windbreak. Additionally, the concentration of young trees in solid sets, with high production of flush season-long is likely to attract ACP at a higher rate than within mature orchards containing randomly interspersed resets. It has been demonstrated that the proportion of ACP immigrating into a grove was correlated to the flush production of citrus trees. Overall our two experiments tend to demonstrate that microclimate and grove complexity are likely to influence ACP population densities. In the context of integrated pest management for ACP and associated HLB, our results advocate for conservation of windbreaks and hedgerows and support the implementation of new ones.
During this funding period, we investigated the levels of resistance in field populations of the Asian citrus psyllid (ACP) in commercial groves across central and southern Florida to six classes of insecticides and compared those results to previous years. In addition, we determined the baseline levels to one new chemistry, flupyradifurone, a butenolide agonist that acts on the insect nicotinic acetylcholine receptor, the same target as the neonicotinoids, imidacloprid, thiamethoxam and clothianidin. In years 2009 ‘ 2012, a decrease in susceptibility to several major classes of insecticides was observed in field ACP. In contrast, surveys conducted in 2013 and 2014 showed the return of the LD50 response in field psyllids to be equal to that of the reference laboratory strain. However, examination of the probit lines from the dose-response of field psyllids compared to laboratory strain, and with one another, revealed significant differences in response to insecticidal treatment for several populations for a number of the insecticides tested. In these studies, and those of prior funding periods, four fundamental determinations were made: (1) the enzymatic profile of detoxifying enzymes of ACP is diverse, robust and responsive. It is well documented that the evolutionary dynamic of plant-feeding insects in response to allelochemicals produced by their host plants has led to diverse enzymatic systems by the insects to detoxify the protective toxins produced by plants, and ACP appears to be no exception, (2) the insecticidal response of ACP is dynamic, showing potential for resistance against all major classes of insecticides currently used to control this insect pest, and that insecticidal response can return to basal levels based on LD estimates, (3) based on the analysis of probit lines, it appears that differences between populations of ACP exist at the genetic/enzymatic levels, potentially leaving some populations ‘primed’ for resistance with pressure from insecticidal exposure and (4) an LD estimate, whether it be LD50, LD75, or LD95, is probably not an appropriate metric alone to monitor for resistance in field populations of ACP. This last finding is of particular importance because as was discovered in these studies, differences in insecticidal response between populations in the last two years were only apparent by probit analysis. Given how quickly ACP can develop resistance, it is imperative to be using the most sensitive method for detection as possible. Because we did not find resistant populations during this funding period in which to study the biochemical mechanisms underlying resistance, three attempts were made to develop resistant colonies in-house with no success. However, given that the resistance induced in laboratory colonies is typically polygenetic, whereas the resistance observed in field populations is usually monogenetic, it is unlikely that the mechanisms induced in laboratory insects would be representative of the mechanism that would be observed in the field. Instead of continuing to attempt to develop a resistant colony to study insecticide resistant mechanisms in ACP, we concentrated on characterizing the voltage-gated sodium channel, the target of pyrethroids. This channel is well-known for target-site mutations that result in reduced binding of the insecticide to its target in a wide diversity of insects. We found that ACP is no exception, and that there is potential for the target to become insensitive to pyrethroids through known mutations. In particular, ACP has potential for two particular single-nucleotide polymorphisms that would result in target-site insensitivity for two of the most effective kdr mutations known. Given the wide-spread nature of these mutations within insect species, including other Hemipterans, we conclude that pressure from pyrethroids would result in target-site insensitivity, resulting in the reduced efficacy of this important insecticidal chemistry.
March 26, 2015 The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. Guignardia citricarpa, the causal agent of citrus black spot, is a newly emerging disease of citrus in Florida. The disease is concentrated in Southwest Florida. It has not been clear when the inoculum was being formed or where in the tree. The mating type structure of G. citricarpa was shown to be heterothallic unlike the common citrus endophyte G. mangiferae, which is homothallic. Thus far, it was shown that G. citricarpa conidia germinate, form appressoria, and penetrate leaves through the culticle. No further changes have been observed over several months and no symptoms have been observed over 10 months. When Diaporthe citri and G. citricarpa are co-inoculated on twigs, D. citri out competes in suboptimal growing conditions for G. citricarpa. The optimal relative humidity range for production of conidia at 24’C was between 82 and 100% but some detection of G. citricarpa DNA was possible at the 43 and 72% RH treatment.
March 2015 The objectives of this proposal are 1) to determine if a) leaf litter biodegradation treatments reduce Guignardia spp. pseudothecia and improve control afforded by routine fungicide applications; b) if biodegradation is affected by the current fungicide application practices; and c) whether the biodegradation treatments will affect current citrus best management practices (BMP); 2) to determine the seasonal dynamics of leaf litter inoculum load in varying management regime intensities and how environment affects pseudothecia production in the leaf litter; 3.) to test if the resistance to black spot in the leaves and fruit in sour orange is correlated and under simple genetic control through laboratory and field testing of progeny of sour orange crosses in both Florida and Australia. The small plot work of the leaf litter was completed and the data processing has begun. The molecular data is nearly complete and processing has begun. We are waiting for symptom expression to take data on the large field trial. To look at the effect of bagasse, a controlled experiment portion was conducted twice. It involved an in vitro study of the decomposition of citrus leaves and inactivation of G. citricarpa by various amendments including bagasse including the microbial consortium of fungal and bacterial strains to aid in the decomposition of bagasse and of citrus leaves. A field experiment at Citra was conducted in addition and leaf decomposition was observed for 4 months with collections once a month. Two factors contributed to greater leaf decomposition; bagasse mulch and soil moisture (distance from microjet sprinklers). A manuscript is in preparation. Collection of leaf litter material continues in Florida and is being collected every two weeks from a grove with moderate black spot incidence in the previous season. Some data summary has begun and we are finding much lower structure numbers and incidence in the leaf litter. This is likely because the disease pressure is much lower and more scattered in Florida than it has been in Australia historically. We expect to find the same trends but since our climate is very wet, there may be differences in leaf litter cycle. Preliminary data analysis is underway. Phyllosticta capitalensis and G. citricarpa isolates were collected in Australia to look at the competitive interaction of the two fungi. A preliminary experiment was run and there appeared to be no inhibitory effect of the two fungi but it was decided to modify the technique and the second experiment is underway. In comparing the fungicide sensitivity of G. citricarpa and P. capitalensis a full experiment was undertaken. Some of the EC50’s have been calculated and data analysis is on going. Leaf litter collection is on-going in the Central Burnett district in three orchards. There is also a fungicide trial on-going with staggered protectant fungicides that will hopefully identify key infection events. So far pycnidia and spermogonia are the only types of Guignardia spp. structures observed in the leaf litter. Germplasm has been inoculated and we have begun the long wait for symptoms. A parallel microplot experiment for leaf litter decomposition to that in Florida has been initiated in Queensland but no results are ready to be reported.
Foot rot, gummosis, and root rot caused by Phytophthora nicotianae are widespread diseases in citrus groves in South Texas. In order to control the spread and disease development we proposed to (1) identify potential inoculum sources of P. nicotianae within the citrus production system in South Texas, and (2) to determine the optimal timing of field application of chemical control agents against Phytophthora infections utilizing different irrigation systems. For objective one, we started a survey of citrus nursery facilities and of different components of citrus grove operations in the Lower Rio Grande Valley (LRGV) for the presence of P. nicotianae. Samples from different potential inoculum sources including protective sponge wrappings around mature trees, leaf litter found throughout groves as well as soil and irrigation water sources from nurseries were assessed for P. nicotianae using standard practices. Observations were also taken of other fungal pathogens recovered from the same set of samples. Results of samples collected from one orchard during February 2015 revealed the presence of Fusarium and Pythium from protective sponge, soil and leaf litter. At this time of the year the recovery of P. nicotianae in protective sponge and leaves was very difficult and only low propagules were recovered from the soil. In addition, we tested soil, roots and water from one citrus nursery for the presence of P. nicotianae. Only Fusarium and Pythium were recovered from nursery materials, suggesting that inoculum of P. nicotianae in LRGV groves perpetuate within citrus groves and are unlikely to originate from the current nursery system. To identify P. nicotianae sources of inoculum, during the spring of 2015 we will continue the survey to sample soil, water and plant materials from mature groves. To attain objective two, we have trained a Master’s student for the isolation and quantification of P. nicotianae from the soil. In addition, we have in stock the chemicals that will be used for the study. We also determined the site for the study, where we will be testing the timing of chemical application in flood, micro sprinkler jets and drip irrigation systems for the optimal control of P. nicotianae in mature groves. P. nicotianae propagules will be assessed before the spring application from all three irrigation systems and monitored throughout the study.
The Citrus Flowering Monitor system predicted a February 20 bloom date for round oranges for spring 2015. Based on this predicted timing, flowering branches were selected for data collection in 4 Hamlin, 4 Valencia, two Murcott and two Sunburst locations. Three trees at each site were selected with branch units on each side of the tree row. Vegetative and flowering buds will be tracked for leaf and flower numbers and developmental stages. Although some bloom was evident in January, no significant off-season bloom related to HLB stresses were detected at that time. Restructuring the monitor system is now underway.
The Citrus Flowering Monitor system predicted a February 20 bloom date for round oranges for spring 2015. Based on this predicted timing, flowering branches were selected for data collection in 4 Hamlin, 4 Valencia, two Murcott and two Sunburst locations. Three trees at each site were selected with branch units on each side of the tree row. Vegetative and flowering buds will be tracked for leaf and flower numbers and developmental stages. Although some bloom was evident in January, no significant off-season bloom related to HLB stresses were detected at that time. Restructuring the monitor system is now underway.
December 2014 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: Manuscript preparation continues for the DMI fungicides. Assays with SDHI’s are continuing and different media are being evaluated for best functionality. Preliminary experiments with Cannonball and Vanguard have begun. Objective 2: Trial data were collected and analysis is underway. Preparation for a citrus industry article is underway. Objective 3: Data was collected from Groves II (emerging disease) and III (intermediate disease) and the data is being compiled. Objective 4: Valencia fruit were collected from a symptomatic grove for further experiments. We also looked at the effect of hot water (53, 56, 59, 62C) at different exposure times of (0, 10, 20, 30, 60, 120, 180, 300s) on mycelial growth. All were compared to 25C. Very little growth inhibition occurred at 53C only 30 s of exposure to 62C caused 100% inhibition. When tried on asymptomatic fruit, no hot water treatment at any exposure time gave significantly (P<0.05) different incidences of disease from a dry control. Similar results were seen for symptomatic fruit. There was small but significant reduction (P<0.05) in severity when the asymptomatic fruit were treated at 56C for 120s.
December 2014 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: Tests with with the DMIs are completed with assays with a QoI/DMI on-going. Molecular characterization of CYP51 was completed and there were no unusual features. Manuscript preparation continues. Assays with SDHI’s have been initiated. Objective 2: We are waiting for symptoms to be expressed to collect the trial data. Objective 3: Preliminary analysis has been carried out on Groves I through III. Where Grove I shows total infectivity of the rows scouted, clustering analysis cannot be done. In Grove with emerging disease (Grove II) and intermediate grove (Grove III) both show clustering according to spatial analysis in R (Ripley’s K, Binomial distribution etc’). We are still waiting for color change in fruits to re-map Grove II and III for assessment of spread, and severity. Some initial scouting has begun but symptom expression is not yet at a high enough intensity to get a good estimate of disease intensity. Objective 4: Two sets of in vivo experiments were conducted using large numbers of naturally infected fruit. Fresh Valencia oranges from a block infected with citrus black spot were harvested and evaluated for presence/absence of CBS lesions and equal numbers of randomized fruit were distributed among the different treatments. Fruit were treated with one of seven fungicides at ambient or heated (10 or 13’C) temperatures. The fungicides include azoxystrobin, fludioxonil, pyrimethanil, phosphorous acid, Imazalil, thiabendazole, and sodium-o-phenylphenate. Each of these have formulations that are registered for postharvest use on citrus. Control fruit were either left dry or dipped in water alone. After treatments, the fruits were air dried and kept at 25’C, 70% relative humidity, 3 ppm ethylene, and continuous light condition to promote CBS lesion development. In further analysis of the data it was found that fungicide had a significant effect (P > 0.05) with TBZ having the lowest number of lesions. Neither the solution temperature nor the solution temperature and fungicide interaction had a significant effect on lesion number or percent incidence for asymptomatic fruit. There was a lot of variation among fruit that might have masked effects. There were no significant differences among treatments for symptomatic fruit as expected.
Growers apply insecticides to control ACP as a tactic to prevent or reduce the incidence of HLB but with little or no success. Plant resistance to colonization by ACP populations has been reported in the genotype Poncirus trifoliata L., which is cross compatible with citrus. The Citrus Research Center (CRC) in Riverside, California, maintains a large number of accessions of P. trifoliata. We conducted four free-choice experiments with a total of 29 CRC accessions of P. trifoliata and found that 19 were colonized by fewer eggs than a susceptible sweet-orange cultivar. Subsequent infestation densities of nymphs generally reflected reductions in oviposition but there were notable exceptions ‘ some accessions may be susceptible to oviposition but contain traits that confer antibiosis to nymphs. In no-choice experiments with seedling plants representing eight P. trifoliata accessions, oviposition rates were reduced as compared to sweet orange and it appeared that factors associated with mature leaves were involved. In no-choice experiments with adult ACP confined to flush cuttings, large reductions were observed in numbers of eggs and nymphs on each of five P. trifoliata accessions. Based on these results in conjunction with published reports, a number of accessions of P. trifoliata have exhibited resistance to ACP colonization. Accessions that have consistently shown resistance to oviposition include CRC 2554, 3206, 3207, 3209, 3218, 3338, 3412, 3486, 3547, 3548, 3588, and 3882, The level of resistance has been variable possibly due to environmental conditions, plant age, proximity of plants to susceptible germplasm, and other factors. We continue to study the behavior of ACP adults in choice and no-choice arenas on Citrus macrophylla and Poncirus trifoliata genotypes. Electronic penetration graph (EPG) studies suggest that ACP adults placed on resistant P. trifoliata genotypes spend significantly more time ingesting from xylem and less time ingesting from phloem vascular elements. Work by Dr. El-Desouky Ammar working in our lab and recently published (PLOS ONE 2014 9(10): e10919) suggests that differences in the internal distribution of sclerenchyma cells may inhibit stylet passage resulting in fewer successful feeding bouts from the phloem. Our EPG results support this hypothesis. Data from oviposition studies are being analyzed and will be presented in the next progress report.
We transiently expressed in plants artificial microRNAs (amiRNAs) targeting the V-ATPase mRNA of Bactericera cockerelli. We used agroinfection-compatible Tobacco mosaic virus (TMV) and Tomato mottle virus (ToMoV-A, TAV) expression vectors, and a virus-free 35S promoter driven binary vector and delivered amiRNAs through transient expression in plants and through artificial diets. For the in planta transient expression, the viral vectors, TMV (RNA viral vector) and TAV (DNA viral vector), and non-viral vector carrying amiRNA precursors were expressed via agro-infiltration in Nicotiana tabacum. We harvested small RNAs from tissues and re-sequenced products from these plants to ensure the specificity of amiRNA production. Bioinformatic analyses are underway. For artificial diet feeding experiments, we expressed amiRNAs via agro-infiltration in N. benthamiana plants and the processed amiRNAs were extracted from the infiltrated tissues and mixed with 20% sucrose for feeding tests. We evaluated the B. cockerelli from the feeding tests by mortality assays and reverse transcription (RT)-quantitative PCR (RT-qPCR) for target mRNA expression levels. Our results from the RT-qPCR were compared with the results of small RNA Northern blot and Hi-Seq illumina deep sequencing analysis. In single B. cockerelli-analysis of our initial plant feeding tests, the B. cockerelli fed on tissues expressing high amounts of the amiRNAs of target mRNA showed high variation of target gene expression levels versus more stable expression levels seen in controls. This variation could due to the distribution of amiRNAs in plant tissues and/or the areas where B. cockerelli fed. We are now developing transgenic plants expressing amiRNAs for further biological analysis.
The goal of this project was to test the effectiveness of the entomopathogenic fungus Isaria fumosorosea (Pfr 97) as a biological control tool for the Asian citrus psyllid (ACP) to complement ongoing biological control with the nymphal parasitoid Tamarixia radiata. adults can transmit the spores to existing populations of psyllids. The vertical transmission study was conducted in October-November 2013 at a residential trailer park in Weslaco. For this study, we used laboratory-reared psyllids that were regularly tested for Candidatus Liberibacter asiaticus (CLas) and confirmed CLas free. A group of 500 psyllids was collected from the lab-colony into a 25-ml tube and dusted with the fungus by adding 10 mg of Pfr 97 and gently shaking the tube for 10 seconds. Twenty adults ACP were aspirated in a glass vial and enclosed in a sleeve cage attached to a branch of mature grapefruit trees for a total of 10 replications per treatment. The four treatments tested were 1) flushing shoots infested with dusted ACP, 2) flushing shoots infested with healthy ACP, 3) mature shoots infested with dusted ACP and 4) mature shoots infested with healthy ACP. In the second study conducted in March-June 2014, ACP-infested flush shoots were selected from the same residential areas from lemon trees, and enclosed in sleeve cages. The initial number of adults and nymphs found feeding on these psyllids was counted in situ. Ten Pfr97-dusted psyllids were introduced in the sleeve cages of 10 previously infested flush shoots. Ten other flush shoots serving as control received 10 healthy psyllids. A sample of 20 adult psyllids per flush shoot was collected to determine their Isaria-infection status one week after introducing psyllids in the sleeve cages. One month later, the sleeve cages were removed and the total numbers of psyllid recovered tallied. In the vertical transmission study, mortality of healthy psyllids introduced after the Pfr97-infected psyllids ranged from 6 to 38%, and varied with flush shoot stage and prior contact of shoot with Isaria. Mortality was highest on mature shoots that were previously infested with Isaria-dusted psyllids (38%), while the lowest morality of 6% was recorded on control-young shoots. Prior exposure of flush shoots to Isaria-dusted psyllids increased adult ACP mortality by 17% on young flush shoots and by 26% on mature flush shoots. Three weeks after removal of adult psyllids, no additional psyllid was recovered from nature shoots and only few ACP adults and nymphs were recovered from shoots that were juvenile and expanding at the initiation of the trial. Only a subsample of 18 adults per treatment with young shoot were plated. About 39% (7 out of 18) of psyllids plated were infected with Isaria versus no infected psyllid in the control. These results suggest that Isaria-dusted adult psyllids that colonized a flush can transmit the entomopathogen to the next generation of psyllids and that vertical transmission occurs with Isaria. IIn the second study we observed that 72% of adult psyllids plated from flush shoots that were infested with Isaria-dusted psyllids were infected compared to only 3% in the control treatment. These results suggest that adding Isaria-dusted psyllids to established colonies of psyllids lead to infection of their congeners established on shoots. In fact, of the 20 adult ACP palted for Isaria infection, a maximum of 50% (10 dusted introduced) was expected to be infected, but there was an additional number of psyllids infected that could only come from lateral transmission of from infected individuals introduced in the sleeve cages. The total number of adult psyllids produced from flush shoots exposed to Pfr 97-inoculated ACP was reduced by 21% relative to the untreated control.
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