Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are valuable for monitoring CLM but need further evaluation to optimize use for management. The objectives of the proposed project include: (1) assess species composition of trap catches, (2) optimize spatial and temporal distribution of pheromone traps. Laboratory Technician, Vincent Ficarrotta has continued to extract DNA from P. citrella samples. An additional 196 samples have been sequenced for COI barcoding to target the 658 bp region of COI gene. We recently switched extraction methods from QIAGEN DNA Blood Tissue Kit to OmniPrep. The dataset was merged with our previously published genetic dataset (Kawahara et al. 2013) generated from this project. The OK Slough had the highest number of samples sequenced, and within these samples, P. citrella was the most common, followed by P. vitegenella. Other species, such as the two new Phyllocnistis species, are lured to traps at a low frequency (’11 specimens/year) in all years sampled thus far. While the numbers show strong bias towards P. citrella, it is clear that in all years, multiple species of Phyllocnistis readily come to CLM lures, following the initial finding of Kawahara et al. (2013). We also generated a new ML tree with additional samples analyzed on UF’s High Performance Computing Cluster (HPC). This tree shows largely similar results as Kawahara et al. (2013), where the majority of samples obtained grouped with already-known specimens of P. citrella. Bootstrap values for clades were generally low for most groups (<50%), which is comparable to prior results based on a single marker (e.g., DePrins and Kawahara [2012]). However, there were some notable results, such as the strong sister group relationship of P. citrella (North American) to P. citrella (Asia). Further, there is strong support for the monophyly of P. citrella (NA and Asia), P. insignis, and Phyllocnistis new sp. 1. Other taxa that were included in the dataset, but not generated from this particular project, also show strong bootstrap support for their monophyly, which will be relevant to future studies on non - P. citrella related species in the genus (e.g., P. hyperpersea (100% BP), P. longipalpus (100% BP), P. perseafolia (100% BP), P. populiella (93% BP), P. saligna (100% BP)). We also see relatively strong genetic distance between each group (>2-4% in many cases), supporting the hypothesis that these clades should be treated as separate species. Finally, 212 sequences of the 658 bp COI barcode region that were recently sequenced were submitted to GenBank (www.ncbi.org) as proposed in the original project proposal. The GenBank numbers are being acquired and verified by NCBI staff. We are awaiting results of review and a decision on “Placement Density and Longevity of Pheromone Traps for Monitoring of Citrus Leafminer, Phyllocnistis citrella (Lepidoptera: Gracillariidae)” by P. Vanaclocha, M. M. Jones, C. Monz’ and P. A. Stansly. Mark-release-recapture work using fluorescent powders has indicated that male moths are capable of flying 140 m in 24 h. We determined that release immediately after application of powders is essential to capturing marked moths. We have replicated traps at the distances over four release dates and are preparing to deploy traps at 280 m and 420 m from the release site. We are also preparing to sample Rb-spiked citrus to evaluate Rb uptake and detection in CLM for mark-recapture applications.
Previously, we have reported that acquisition and transmission of Las are less efficient at temperatures below 22oC and above 27oC, and this effect also occurs when psyllids are pre-exposed to temperature for 2 or 4 days prior to acquisition experiments. Observations of cuticular melanization in psyllids exposed to extreme temperatures suggest that this process may be associated with corresponding reductions in acquisition and transmission efficiency. Seven target genes from five gene families (rhodopsin, bursicon, Yellow C, DcAWD, and multi-copper oxidase) have been identified to evaluate how temperature-mediated gene expression influence Las transmission (objective 1). Quantitative polymerase chain reaction (qPCR) assays were developed for each of the target genes, and the relative expression of each gene among treatments was compared using the 2-..CT method by normalizing to psyllid wg gene expression, followed by normalization to the treatment based on the lowest gene expression. Subsequently, we have identified changes in the relative expression of four genes from this group in association with melanization. Currently, experiments are underway to evaluate temperature mediate changes in expression of these genes, and their correlation with melanization and Las transmission. Whole plant experiments are ongoing, but initial results suggest that that down regulation of at least one gene is associated with reduced acquisition at 16oC compared with 27oC. We will further evaluate the role of this gene in Las acquisition using dsRNA. Establishment, maintenance, and characterization of psyllid isolines from Florida is ongoing (objective 3). We have identified one isoline in particular that exhibits increased egg laying compared with other populations. Currently, we are developing a restriction enzyme-base assay to rapidly screen for Wolbachia strains in these isolines to determine their association with reproductive fitness. An additional component of this project is germ-line transformation of psyllids. Optimization of egg injections is ongoing. Survival of nymphs following egg injections in Petri dishes was low. Successful egg hatches were obtained using an artificial diet as an alternative approach, but was not significantly greater than the petri dishes procedure.
The goal of this research has been to monitor insecticide susceptibility in Florida populations of Asian citrus psyllid (ACP) and to determine the molecular mechanisms of resistance. We have completed our investigation of the resistance mechanisms to pyrethroids in ACP. The voltage-gated sodium channel (VGSC) is the primary target of pyrethroids, and is prone to mutations which render pyrethroids ineffective through target-site insensitivity. Mutations to the channel have been reported in a wide diversity of arthropod species. We characterized the VGSC of ACP to determine the potential of the channel to develop similar mutations. Pyrethroids are an important component of management rotations for this pest species and their loss would significantly hinder control efforts. The VGSC in ACP shows as much potential for alternative splicing as any other characterized insect, including variants which have been shown to have reduced pyrethroid sensitivity. Furthermore, it shows significant potential to develop target-site insensitivity through kdr mutations, with the greatest potential at kdr position M918. Previous studies demonstrated that ACP has an exquisite enzymatic arsenal to detoxify insecticides resulting in reduced efficacy. The results from this study demonstrate that target-site insensitivity is also a probable basis for insecticide resistance to pyrethroids in this insect species. The VGSC sequence and its molecular characterization should facilitate early elucidation of the underlying cause of an established case of resistance if it should occur. We have evaluated insecticide resistance in field populations of Asian citrus psyllid (ACP) for approximately the past 7 years. This has allowed us to determine the fluctuation of resistance in Florida ACP populations, develop and recommend appropriate rotation schedules, and determine the specific mechanisms mediating resistance in ACP populations. We have already documented reduced susceptibility in regional Florida ACP populations, where prescribed MOAs sometimes are applied up to 12 times/yr to suppress re-infection of trees. By 2011, there was a measurable reduction of insecticide susceptibility among all ACP populations that we examined across the state of Florida. Also, there was a trend for increased resistance from 2009 to 2012. The highest levels of decreased susceptibility found in field and laboratory in Florida ACP varied between 35 to 100 fold. However, at no point was this reduction of susceptibility sufficiently high so as to result in product failure in commercial groves. We estimate that approximately a 100-fold reduction in susceptibility may be associated with product failure based on our research in non-commercial citrus and the laboratory. The coordination of treatments for ACP is meant to enhance insecticide use by preventing rapid re-infestation by psyllids from non-sprayed areas. By the end of 2010, there were 10 CHMAs established. Our 2010 survey was conducted during that inception period of CHMAs. Insecticide resistance was beginning to be a problem at that time. From 2013 to 2014, there has been a progressive drop in resistance observed among ACP populations in Florida, according to our surveys. In fact, based on our sampling capabilities, it appears that since 2013 we have returned to pre-2009 levels of ACP insecticide susceptibility across the state. The number of active CHMAs has risen from the initial 10 to a current 52. We can hope that coordination and effective rotations of insecticides for ACP have contributed to this decline in resistance. However, given that we know that up to 4,000-fold resistance to the very important neonicotinoid insecticides can occur in ACP populations (V zquez-Garc a et al. 2013), we must remain vigilant by rotating MOAs and monitoring resistance.
The main objective of this research was to determine both abiotic and biotic factors that regulate Asian citrus psyllid (ACP) acceptance of plants and pathogen transmission. The goal was to use this information to interfere with the vector’s capability of transmitting pathogen between citrus trees. During the last quarter we have completed our research and data analysis of our wind-break and replanting investigations. We present two orchard experiments where the densities of ACP were compared depending on: (1) the presence or absence of a windbreak and (2) if the groves consisted of a solid set re-planting or an grove with a mixture of mature and reset-replacement trees. (1) Psyllid abundance was measured on the edges of five groves. The factor investigated was the presence or absence of a windbreak. 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. More psyllids were found in the solid set re-plantings as compared with on the resets within mature groves. To our knowledge, this is the first report to demonstrate that the planting strategy of new trees in groves may impact the populations of a horticultural pest. 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. In 4 groves, we sampled the citrus canopy at 3 different heights and at the 4 cardinal directions. We also investigated the potential for alternative habitats for ACP and sampled non-crop vegetation and potential alternative hosts over 2 seasons. We did not find a consistent pattern in the distribution of ACP during winter in citrus, with one exception; canopies facing the southern cardinal direction harbored more ACP than those facing north. We also found that the proportion of psyllids with the green-blue morphotype (the morph with greatest dispersal capability and likely the major driver of spring grove infestations) increased following insecticide application and after cold weather. Finally, we investigated the potential existence of alternative hosts on which ACP could feed and/or reproduce in the absence of young emerging citrus leaves during winter. We did not find evidence of an alternative host during winter for ACP in central Florida.
The large-scale validation of citrus leafminer (CLM) disruption with the ISCA DCEPT CLM technology has continued. During this past quarter we have applied our 2015 application of the treatment. The deployment of the treatment was successful. Currently we are monitoring populations of CLM in treated and control plots, as well as, damage incidence. Male moth activity is being monitored with pheromone traps that were deployed after treatments were applied. Damage is being monitored by assessing CLM infestation of new leaves.
This is a continuation of the previous project entitled ‘Screening effective chemical compounds against citrus HLB bacterium Candidatus Liberibacter’ (Project # Contest 400). The aims of this project is to rapidly screen the potential chemical compounds for the control of citrus HLB using a graft-based screening method, then to further evaluate the selected compounds in the greenhouse and field by bark-application, trunk-injection and/or root-uptake. Based on the rank of an expert panel and the action mode of the compound, 102 compounds from the Contest were tested using graft-based screening methods. These compounds were classified into four groups based on Ct value in the inoculated plants, scion infected percentage, Las transmission percentage, and the scion-survival percentage and the scion-growth percentage, i.e. (i) Highly effective (8.9 %); (ii) Effective (24.5 %); (iii) partly effective (21.5 %); (iv) Non-effective (45.0 %). A total of 144 treatments from 24 effective antimicrobials or their combinations, three temperatures (40 ‘C, 42 ‘C and 45’C), and two applications (bark-application and root-uptake) were carried out with three replicates in the greenhouse. Six effective treatments was applied in the field by gravity bag infusion. Some treatments were not effective against citrus HLB in the field because of solubility and/or vascular transport issues. The field trial will continue and merge with our integrated control strategy project(CRDF#910).
This is the first year of a 3-year project. The overall project is focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. Based on efficiency of eliminating Las bacterium and phytotoxicity of chemical compounds, seven effective antimicrobial compounds (Amp, ZS,Va, Pcy, Carv, SD and Al(OH)3) screened by graft-based assay were applied on HLB-affected citrus trees by gravity bag infusion. The preliminary results showed that the Las bacterium was reduced significantly by Amp and SD treatment at 10 month after initial treatment. The control trees still had high Las bacterial titers. Some compounds, such as Al(OH)3, ZS and Va, were difficult to be dissolve in water, easily blocked the injection tubes, and resulted in less uptake by citrus. So in next quarter, we will try to apply these antimicrobials by foliar spray using water in oil nano formulation. In the last quarter, Pcy and Carv were prepared in nanoemulsion and applied on the HLB-affected citrus. The preliminary results showed more and longer new branches in these treatments. In this quarter (January 2015 to April 2015), we tried to improve the application techniques. Nine hundred ml of antimicrobial solutions were absorbed in 48 hr. In the next quarter, we will continue to apply the chemical compounds to citrus trees by foliar spray or trunk-injection coupled with the thermotherapy, and monitor variation of Las bacterium in the citrus tree. Furthermore, thermotherapy and nutrient therapy will be conducted in the future.
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.
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