Sept 30 2012, The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. Trees are finally ready for the next stage. Preliminary preparations are underway.
Sept 30 2012 The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus (Las) prevalence in leaf tissue on a grove scale; 2) what are the flushing patterns of citrus and do the flushing patterns affect the prevalence of Las in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Las on a grove scale and how does it compare the results from the citrus trees in the same grove. In 2008 and 2009 Ebert and Rogers demonstrated that the prevalence of Las in the Asian citrus psyllid (ACP) varied seasonally but the pattern between seasons was not consistent. It was suggested that perhaps the reason for the differences between the years related to the flushing patterns of citrus and the prevalence of the bacterium in the leaves where ACPs are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and ACPs. Psyllid, phenology and plant sample collection has continued through the summer. Processing of samples for PCR continues but still at a slow pace.
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. The graduate student, Nan-Yi Wang, whose Ph.D. project this is, continues his studies in Gainesville this term and is making good progress in his classes. He is also conducting research while in Gainesville. Because the strategy of finding the mating genes via primer homology with related fungi failed, we did a draft sequence of a G. citricarpa isolate. We are analyzing and assembling the sequence currently. We found one border of the known mating gene cluster but we are looking for other border in the remaining contigs. We now have several GFP-transformed isolates that are being evaluated for genetic purity. We are doing repeated selection for genetic purity. Fungi have more than one nuclei per cell and not all may have GFP integrated so we are selecting for spores expressing the protein. The trees have arrived for greenhouse experiment, have been trimmed back and preliminary experiments are in the planning stage. The greenhouse air conditioner has finally been repaired.
Males and females of the Asian Citrus Psyllid, Diaphorina citri, the vector of the citrus greening disease Huanglongbing, locate and communicate with each other using plant-borne vibrations (Wenninger et al., Ann. Ent. Soc. Am 102: 547-555, 2009). The male emits a vibrational ‘call’ when he begins to search for a female. A nearby female may respond with a vibrational ‘reply’ within about a half-second, which alerts the male to move towards her and call again. They continue to ‘duet’ until the male finds her and they mate. The goal of this project is to use these vibrational communication calls to develop a trap that attracts male psyllids searching for females on the tree branches. In the first steps towards trap development, a series of bioassays was conducted in which calls recorded from different males were played back to multiple females using a minishaker attached to the small trees on which they were feeding. The females replied to some male calls more readily than others. The goal of these experiments was to determine a range of signal levels and distances over which the vibrations were active. In other bioassays, calls recorded from replying females were played back to males. Typically, the males moved towards the source of the female replies and at intervals called to elicit further replies. These experiments were conducted to determine the range of attractiveness of signals that mimicked female replies. A series of synthetic calls has been prepared from playback calls and replies that elicited the highest levels of responsiveness, and testing is in progress to establish whether these signals can be played back by small computer systems which could be incorporated into traps. In many cases, the synthetic calls can be produced more easily than the playbacks of real calls. We are also investigating the least expensive ways to produce attractive signals effectively, and the least expensive ways to trap the psyllids once they are attracted.
A novel psyllid trap has been developed in several prototypes that capture Asian citrus psyllids (ACP) and preserves them and their entire contents (genome and that of associates) immediately and for perpetuity. This proposal aims to continue the improvement of the trap and to use the trap to gather new information on the behavior, biology, population dynamics and biological control of ACP/Candidatus Liberibacter asiaticus. Lab and field testing was and continues to be conducted to increase trap efficiency by discovering, manipulating and exploiting unique vector behaviors in response to traps. Currently, a number of investigations by other researchers have been completed while others are ongoing, to discover and identify semiochemicals that actively attract or repel ACP. While a number of plant volatiles and ACP-produced compounds have shown their presence or activity in laboratory bioassays, only low level capture increases (less than or equal to a 25% increase in trap captures over unbaited traps) and inconsistent results have been manifested in field bioassays. Positive and consistent results of attraction and capture of ACP (though not in overwhelming numbers) have been demonstrated to sticky traps colored in some hues of green-yellow. To date the visual modality is the only behavioral response reliable enough to attract psyllids. Therefore, we have conducted a large number of field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulating psyllid behavior around the trap or farther away so that they are moved close enough to the trap to perceive it (i.e., increase trap active distance), and 2). through increased understanding and manipulation of psyllid behaviors in response to various physical parameters of the trap affecting vision or olfaction that will lead to higher capture rates. We have a number of positive results from our bioassays with which to attempt to increase trap efficiency but have yet to reach a level of trap capture rate which is a satisfactory representation of ACP populations in the vicinity of the trap. This work is continuing. Once we have an efficient trap, the trap will be deployed throughout Florida to determine the current statewide distribution of psyllids, Candidatus Liberbacter asiaticus and their associates including at selected retail establishments. The areawide intensive sampling will provide a unique data set from which to detect and determine the natural infection rate of adult vectors by known and novel entomopathogens. The genome sampled will provide a benchmark for future research as appropriate and remain available for continued use in posterity.
We collected dead, preserved D. citri psyllids, and psyllid RNA samples from Florida, China, Brazil, and Taiwan and are in the process of collecting more from Pakistan, Argentina, Mexico and more from China. So far we have performed small RNA sequencing on four separate psyllid samples. We obtained an average of 14 million sequences per sample and initial sequence analyses indicate good quality. We are now in the process of performing bioinformatic analysis on these initial samples to determine if our protocols and procedures give good quality sequence data. We are also analyzing the data to identify overlapping, contiguous small RNAs that indicate the presence of D. citri-infecting viruses. We will then design approaches to confirm virus presence in specific psyllid populations and as viruses are discovered we will attempt to transmit them to D. citri using the UC Davis D. citri population. Simultaneously, we will work to engineer candidate viruses for D. citri RNAi studies.
Two 3-year field experiments are being conducted in two commercial orange blocks in Hendry County (southwest Florida). One of the groves is planted with ‘Earlygold’ oranges and the other with ‘Valencia’ oranges. Average HLB incidence estimated in both groves at the beginning of the experiment based on PCR analysis of a random sample of 160 trees was 98% in ‘Earlygold’ and 42% in ‘Valencia’. Experimental design is randomized complete block with 4 replicates and 4 treatments: (1) No insecticide, (2) Calendar applications, in order to drive vector populations close to 0, (3) nominal threshold of 0.2 psyllids per tap, and (4) nominal threshold of 0.7 psyllids per tap. Calendar applications are being applied approximately every month. In October, treatment (2) was sprayed with Fenpyroximate (Portal) at a rate of 4 pts/ac. The first dormant spray was conducted mid December and treatments (2) and (3) were sprayed with Zeta-cypermethrin (Mustang) at a rate of 4.3 oz/ac. In Janurary 2013 treatments (2), (3) and (4) received a second dormant spray with Phosmet (Imidan 70-W) at 1.3 lbs/ac. In the third week of November the ‘Earlygold’ block was harvested and yields, yield increments with respect to the 2010 harvest as well as several fruit quality parameters were measured for each treatment. The values obtained were correlated to ACP adult cumulative numbers obtained by stem tap sampling during the last two seasons. A trend to higher yields was observed in treatment (2) (92.75 ‘ 4.38 kg/tree) but with no statistical differences with respect to the other treatments (1) (83.76 ‘ 7.12 kg/tree), (3) (79.77 ‘ 11.02 kg/tree) and (4) (86.74 ‘ 7.02 kg/tree) (F = 1.4; df = 3, 9; P = 0.306). Neither were differences found among treatments in yield increases from 2010 to 2012 (F = 0.76; df = 3, 9; P = 0.547). However, there was a significant treatment effect on fruit weight (F = 3.13; df = 3, 9; P < 0.05) with heavier fruit in the treatments that received more sprays (141.2 ' 2.2 and 142.4 ' 2.3 g in (2) and (3) respectively) than in those with fewer insecticidal applications (135.3 ' 2.3 and 134.9 ' 2.3 g in (1) and (4) respectively). Total pounds solids per box and the brix-acid ratio values were similar among treatments (F = 0.59; df = 3, 9; P = 0.637) (F = 0.06; df = 3, 9; P = 0.980). Despite the fact that we found significantly lower ACP cumulative numbers in treatment (2) compared to the rest of the treatments (F = 15.62; df = 3, 9; P < 0.0001), no significant correlation was found between cumulative ACP numbers and yield losses (P = 0.371). This result is possibly explained by low ACP populations found in the 'Earlygold' block during 2012 (0.055 ' 0.012, 0.007 ' 0.002, 0.017 ' 0.004 and 0.053 ' 0.008 ACP adults per stem tap in treatments (1), (2), (3) and (4) respectively). According to the results obtained in the 2012 'Earlygold' block harvest, we could say an average of 0.05 adult ACP per tap throughout the season seems not to produce a significant reduction inn yields in the short term, although trends observed make suggest that yield significant reductions could appear in a longer term. In fact, some fruit parameters such as fruit weight were already negatively affected by these ACP levels of infestation. However, juice quality parameters seem not to be affected by these levels of infestation.
Two 3-year field experiments were conducted in two commercial orange blocks in Hendry County (southwest Florida). One of the groves was planted with ‘EarlyGold’ in December 2001 and the other with ‘Valencia’ oranges in 1999. Average HLB incidence estimated in both groves at the beginning of the experiment based on PCR analysis of a random sample of 160 trees was 98% in ‘Earlygold’ and 42% in ‘Valencia’. Experimental design was RCB with 4 replicates and 4 treatments: (1) No insecticide, (2) Calendar applications, in order to drive vector populations close to 0, (3) nominal threshold of 0.2 psyllids per tap, and (4) nominal threshold of 0.7 psyllids per tap. Calendar applications were applied approximately every month. Broad spectrum insecticides were used in winter and at the end of the growing season. More selective products were chosen during the growing season to conserve natural enemies. Asian citrus psyllid (ACP) populations were monitored every 2 weeks by tap sampling for adults and flush inspection for nymphs. Beneficial arthropod faunal abundance was also monitored by tap sampling, direct inspection and vacuum sampling among others. Life table studies on ACP and citrus leafminer were also employed to study potential effects of insecticides on the biotic mortality. Yields were estimated from all fruit in each plot and brix/acid ratio, pounds solids per box, fruit weight and fruit diameter assessed and related to cumulative numbers of ACP adults. In the event of significant treatment effects, an economic analysis was conducted to estimate economic injury levels for ACP. No differences among treatments were found in 2010 ‘Earlygold’ harvest, just 3 months after initiating the experiments (F = 0.72; df = 3, 9; P = 0.55). Harvest yield increments from 2010 to 2011 were significantly greater from trees receiving monthly sprays compared to the untreated control receiving no insecticide applications (LS Means: t = 3.18; df = 9; P = 0.0112). However, the economic study revealed that a price of $2.19/lb would be needed to pay for the additional cost of the calendar sprays given the yield increments estimated from treatment (2). Treatment (3) was the most cost effective, requiring a price of only $1.24/lb to pay for additional costs of the program. No significant treatment effect on ‘Earlygold’ yield was found in 2012 (F = 0.76; df = 3,9; P = 0.55) although highest yields were again observed for treatment (2). No significant yield effect was found the 2 years in the ‘Valencia’ block in 2010 (F = 0.48; df = 3,9; P = 0.70) or 2011 (F = 0.32; df = 3,9; P = 0.81) in contrast to 2012 (F = 5.25; df = 3,9; P = 0.03). Yields were highest for treatment (2) (315.3 ‘ 19.4 boxes/acre) followed by (3) (275.4 ‘ 19.7 boxes/acre) and lowest for treatments (1) and (4) (242.6 ‘ 14.6 and 226.9 ‘ 34.7 boxes/acre respectively). Again, treatment (3) was the most cost effective, requiring a price of only $0.32/lb solid to pay additional costs of this treatment. ACP cumulative numbers explained a significant portion of the variation in yield using a rectangular hyperbolic regression (F = 13.14; df = 2, 14; P = 0.0009; Pseudo-R2 = 0.69). The estimated parameters of the fitted equation were A = 32.76 ‘ 11.26 and I = 0.0642 ‘ 0.0435. A cost-benefit analysis using the hyperbolic equation to test which ACP densities balanced out the additional ACP application costs with losses due to ACP indicated mean ACP adults per tap throughout the season required to optimize the insecticide program ranged between 0.001 and 0.11. A spreadsheet was prepared to calculate the mean ACP per tap density throughout the season that would optimize applications cost for given values juice price and fruit quality.
Research targeting the development of a new Asian citrus psyllid (ACP) control strategy based on blocking salivary sheath formation continues to advance. We now have identified two enzymatic pathways that degrade the the salivary sheaths based on compositional knowledge and are moving to test the use of these strategies in plants. We have also finished the initial screening of the peptide library provided by Torrey Pines Institute for Molecular Studies. These results have identified peptide mixtures with activity affecting psylllid sheath formation and psyllid survival. These are being advanced into the second phase where individual peptides are identified. We continue to move ahead to identify defined peptide(s) that have adverse affects on the ACP that will be used to construct a synthetic gene(s) encoding the specific peptide(s). These will be used to construct transgenic plant and for topical application strategies to block ACP feeding processes.
Research targeting the development of a new Asian citrus psyllid (ACP) control strategy based on blocking salivary sheath formation was advanced in two main areas. First, we have developed a novel technique to isolate (to purity), fully intact ACP sheaths having both flange and sheath body as a single complete unit (previously our techniques could only isolate the body portion, excluding the flange and we have shown that they are compositionally different). Also, we are now able to isolate at an estimated >90% efficiency, intact sheaths, whereas previously our sheath harvest yield we estimated at <10% efficiency for partial sheath harvests. By isolating full sheaths, at high yield, we are able to effectively determine the complete structural composition of the sheaths by molecular and chemistry approaches. We are presently substantiating our previous GC, IR, and protein analyses that suggested the sheath structure was primarily composed of carbohydrates with some protein components, using these same techniques with the full sheaths. Also, using a barrage of enzymatic analyses, we have identified single gene products that prevent sheath formation. At present we now can both chemically and biological inhibit the formation of, and degrade, ACP sheaths. Currently, we are moving forward with definitive sheath compositional analyses and to application of sheath inhibitory mechanisms in in vivo systems. Second, using our artificial diet bioassay, we are presently de-convoluting a decapeptide amino acid mixture library developed by Torrey Pines Institute for Molecular Studies. Our diet assay is being used as a vehicle to screen and identify specific peptides responsible for biological activity against the sheath and/or psyllid. At present we have been able to determine certain specific activities associated with specific amino acids at defined positions of these decapeptides. Interestingly, we have been able to show both positive and negative impacts on the psyllid with different position specific amino acids. During the next year, defined peptide(s) will be identified that have adverse affects on the ACP that will be used to construct a synthetic gene(s) encoding the specific peptide(s) for use in transgenic plant construction and for topical application strategies to block ACP feeding processes.
We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. To date, we have cloned sequences from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, we evaluated the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). In this objective, Gateway-based vectors were used to express the selected insect dsRNA (Coo2, GSTS1 and S4e) either constitutively (35S promoter) or in a phloem-specific manner. Our results suggest that the M. persicae-specific dsRNA expressed in planta has a negative effect on both the lifespan of the insects and the number of offspring generated. In the fall of 2010, we began working on objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Previously, we conducted 3′ rapid amplification of cDNA from vacuolar ATP synthase subunit G, S4e, and .-tubulin transcripts from D. citri. We have now inserted sequences of the aforementioned transcripts into Gateway-based vectors downstream of both the constitutive 35S and our novel phloem-specific citrus CsSUS1 promoters. To date, we are in the process of transforming and regenerating citrus lines with the D. citri-specific gateway vectors for evaluation and use by the Florida citrus industry. Currently we have several lines containing gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. We have regenerated at least one line for each of the constructs of interest, and we are in the process of in vitro micro propagation to produce additional copies of each line. We also regenerated additional transformed lines, and beginning to characterize the transgene expression to select lines for micro propagation. We are beginning transformation of additional lines with the other transcript/promoter combinations.
We collected dead, preserved D. citri psyllids, and psyllid RNA samples from Florida, China, Brazil, and Taiwan. We are still attempting to obtain D. citri RNA samples from Pakistan, India, Mexico and Central America. We extracted total RNAs and from these we have isolated the small RNAs which result from virus infections. These have been purified and we prepared small RNA libraries for Illumina next generation small RNA sequence analysis. Samples have been submitted for RNA sequencing and we are waiting for the sequence data. We also have obtained permits and now have started a colony of D. citri within the UC Davis Contained Research Facility. As viruses are discovered we will attempt to transmit them to our D. citri and ultimately engineer them for RNAi studies.
We surveyed the occurrence of volatile compounds in the headspace over six species in the family Rutaceae that differ in their susceptibility to the Asian citrus psyllid. Some compounds were unique and others were common to several species. A manuscript (Robbins et al., “Volatile profiles of young leaves of Rutaceae spp. varying in susceptibility to the Asian citrus psyllid (Hemiptera: Psyllidae)” will appear in the next number of the Florida Entomologist. The list of volatile host plant compounds is being systematically surveyed by electroantennogram (EAG) and coupled gas chromatograph-electroantennogram detection (GC-EAD) We tested volatiles by EAG and GC-EAD identified from the headspace of citrus flush as well as compounds identified from the cuticle of female psyllid. Cuticular compounds were tested against the antennae of both male and female psyllids. In these preliminary studies, none of the heavy weight cuticular compounds were found to stimulate the antennae of either sex. We plan to continue examining other cuticular compounds to test for evidence of activity. We discovered that breakdown products of two common volatiles isolated from the headspace over citrus flush were highly stimulatory to the antennae of both sexes of the Asian citrus psyllid. This is a novel discovery since no degradation products of citrus volatiles have ever been found to stimulate the antennae of the psyllid. We have identified these degradation products by GC-Mass Spectrometry and are now testing them for behavioral activity.
Xambr’ Brazil Plots: Designed to examine the effect of windbreaks, copper sprays to reduce infection, and leafminer treatments to determine their individual and combined effects on control of citrus canker. Windbreaks were completed and plants were be established in Mid April 2010, but severe winds damaged the windbreaks during two storm events. These windbreaks have been reinforced and rebuilt. From July through September 2012, we continued replanting citrus trees damaged by high winds and continued replacing windbreak damaged screens and poles. Windbreak and citrus plants will be of sufficient size to begin the experiment in March 2013. New windbreak trials established in Saint Lucie County. Preliminary disease data was obtained from sampling trees adjacent to the weather stations. Results indicated that disease incidence and severity across the field was related to proximity to the wind breaks, with least disease near the wind breaks. Wind speeds were least near wind breaks. Programmable leaf wetness controller (PLWC) to examine canker bacterial survival in the field. Software was written, debugged, is complete, and the control program is working well. Development of leaf wetness sensors that function across the full range of wetness has eluded scientists for many years. We have designed and constructed sensors based on completely new technology and continue to debug them prior to deployment. During this quarter we have explored the use of polymeric tissue and a conduit, that is water sensitive and change shape when it comes in contact with water or an electric charge. Unfortunately the distortion was quite large and not always in the same direction (dependent on where the water hit). We also tested the current change through the strip between wet and dry and we are currently analyzing the data. Project publications: Bock, C. H., Graham, J. H., Gottwald, T. R., Cook, A. Z., and Parker, P. E. 2010. Wind speed effects on the quantity of Xanthomonas citri subsp. citri dispersed downwind from canopies of grapefruit tree infected with citrus canker. Plant Di Bock C.H., Graham, J.H., Gottwald, T.R., Cook, A.Z., and Parker, P.E. 2010. Wind speed and wind-associated leaf injury affect severity of citrus canker on Swingle citrumelo. Eur J. Plant Path 128:21-38 Bock, CH, Parker, PE, Cook, AZ, Graham, JH and Gottwald, TR. 2001. Infection and decontamination of citrus canker and inoculated the surfaces. Crop Protection 30:259-264. Hall, D.G., Gottwald, T.R. and C.H. Bock. 2010. Exacerbation of citrus canker by citrus leafminer Phyllocnistis citrella in Florida. Florida Entomologist. Florida Entomologist 93:558-566. Bock, C.H., Gottwald, T.R. and Parker, P.E. 2011. Distribution of canker lesions on the surface of diseased grapefruit. Plant Pathology (Accepted). Bock, C.H., Cook, A.Z., Parker, P.E., Gottwald, T.R., and Graham, J.H. 2011. Some characteristics of the dispersal plume of bacteria of Xanthomonas citri subsp citri in wind-driven splash downwind of canker-infected grapefruit tree canopies. (Plant Disease). Bock C.H., Graham J.H., Cook A. Z., Parker P.E., and Gottwald T.R. 2012. Predisposition of citrus foliage to infection with Xanthomonas citri subsp. citri. (Submitted to Plant Disease).
In this final study, we tested if widely-used neonicotinoid insecticides can prevent Las acquisition and/or inoculation on citrus plants. In a first experiment, we evaluated the effects of insecticide mode of application and citrus branch age on pathogen acquisition by ACP nymphs and adults, and found that both foliar (contact effect) and drench (systemic effect) applications of neonicotinoids were effective in preventing acquisition. The results were described in detail in the previous report. Recently, a second experiment was carried out to test if the same insecticide treatments (foliar and drench applications of neonicotinoids) can prevent Las inoculation by ACP on citrus nursery trees. Healthy sweet orange nursery trees [Citrus sinensis (L.) Osbeck grafted on ‘Rangpur’ lime] with or without young shoots were submitted to the following treatments: a) foliar application of imidacloprid (200 SC ‘ 0.24 ml c.p/1 L water), for contact action; b) drench application of thiametoxam (250 WG ‘ 1 g c.p./plant, diluted in 50 ml water), for systemic action; and c) untreated plants (water only; positive control for estimating inoculation efficiency after exposure to infective psyllids). Each of these test plants were exposed to 10 psyllid adults that had been previously submitted as 4th instar nymphs to a 10-day AAP on Las-infected plants, followed by 10 days on intermediate citrus healthy seedlings to conclude the latency period. The groups of 10 psyllids were confined inside clear plastic cup cages on a single apical branch (young or mature) of the test plants for a 72-h IAP. The mortality rate on test plants of each insecticide treatment was recorded at 1, 3, 24, 48 and 72 h after the beginning of the IAP. The experiment was repeated three times, using different source plants and insects for Las acquisition. Each trial was considered as a block in a randomized block design, with 6 treatments and 5 replicates (test plants). The inoculated test plants were evaluated by qPCR up to 12 months after the IAP. Transmission to test plants was detected in all treatments. By pooling the data of the three transmission trials, significant proportions of infected test plants were observed in treatments with systemic (drench application) (13.3%) and contact insecticides (foliar application) (6.7%). The transmissions rate to untreated plants (positive controls) was 26.7 and 13.3% for inoculations in mature and young branches, respectively. Regarding insect mortality, there was no effect of branch age on the effect of insecticide treatments (F = 0.72; P > 0.79). However, there was significant variation in mortality depending on the application mode (F = 10.2; P > 0.0001). The foliar application (contact insecticide) resulted in higher psyllid mortality, with a light knockdown effect (6% mortality) in the first hour after application, increasing to 60, 76, 82 and 84% at 3, 24, 48 and 72 h, respectively. The drench application (systemic action) showed no knockdown effect (F = 10.2; P > 0.30), and caused mortality rates lower than those observed for the foliar application in all evaluations. Except for the first hour (no knockdown effect), the mortality caused by drench application differed from those on the untreated controls, but its control efficiency estimated based on Abbott’s formula was <45% after 72 h. Despite the significant effects on psyllid mortality, the results of these two experiments indicate that the insecticides and application modes tested here do not prevent Las inoculation by D. citri to healthy citrus trees, although they can drastically affect bacterial acquisition.
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