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Asiatic citrus canker (ACC) foliar lesions were evaluated on progenies of 84 seed-source genotypes ("parent genotypes") from the Citrus Variety Collection (CVC) of the University of California at Riverside (UCR) of Citrus trifoliata and hybrids between C. trifoliata and other Citrus species and hybrids. Trees were planted Aug. 2013 in a completely randomized design at the Fort Pierce U.S. Department of Agriculture (USDA) grove. Plants were assessed visually Aug. 2017, Sept. 2019, and Sept. 2020 for distinctive ACC lesion incidence and severity. Progeny were compared by parent genotypes using nonparametric analysis. Incidence of ACC [percentage of leaves displaying symptoms, verified by quantitative polymerase chain reaction (qPCR) to be associated with Xanthomonas citri pv. citri] across parent genotypes ranged from 8% to 80% (mean, 49%) of leaves affected in 2017, from 4% to 58% (mean, 29%) in 2019, and 8% to 46% (mean, 25%) in 2020. In 2017, of 49 C. trifoliata parent genotypes, only four separated from the two highest ACC-incidence statistical categories [Citrus Research Center (CRC) 3345, 3484, 3888, and 4017]. whereas 29 of the 35 C. trifoliata hybrids displayed lower ACC incidence, which separated from the two highest statistical categories. In 2019, of the C. trifoliata, only six separated from the highest ACC-incidence statistical category (CRC 3330, 3484, 3547, 3549, 3876, and 3888), whereas all 35 C. trifoliata hybrids displayed lower ACC incidence and separated from the highest statistical category, and 26 hybrids separated from 18 of the C. trifoliata. In 2020, only three C. trifoliata separated from the highest ACC-incidence statistical category (CRC 2861, 3549, and 3888) and 20 hybrids separated from 18 of the C. trifoliata. By parent genotype, ACC incidence correlated substantially between each pair of the 3 years, with r2 values of 0.39, 0.57, and 0.65. Of 34 hybrids validated, similar numbers had C. trifoliata, grapefruit (C. 3aurantium var. racemosa), and sweet orange (C. 3aurantium var. sinensis) chloroplasts. Chloroplast type affected ACC incidence and severity, but not in a consistent manner. Near-isogenic groups within C. trifoliata, as determined by DNA markers, were associated with some statistically different ACC sensitivity. Overall, hybrids of C. trifoliata with other citrus types displayed markedly reduced ACC sensitivity compared with C. trifoliata, indicating that this trait is readily overcome through breeding. [ABSTRACT FROM AUTHOR]

The U.S. Department of Agriculture citrus scion breeding program is urgently working on developing huanglongbing (HLB; pathogen Candidatus Liberibacter asiaticus)-tolerant cultivars with excellent fruit quality and productivity when HLB-affected. The slow process of assessing new citrus hybrids is a major impediment to delivery of these much-needed cultivars. We generate thousands of hybrids each year, germinate the seedlings, grow them for 2 years in the greenhouse, plant them at high density in a field where the disease HLB is abundant, grow them for 5 to 10 years, and make selections based on tree performance and fruit quality of these HLB-affected trees. Based on promising reports of accelerated citrus growth when grown in a metallized reflective mulch (MRM) system, we tested the hypothesis that the MRM system may accelerate growth and selection of new hybrid seedlings compared with conventional soil culture (CSC). In the MRM system, tree rows are covered with a layer of metallized plastic film and drip irrigation is installed beneath the plastic. In 2 years of analysis, tree canopy volume was significantly greater with MRM in 2020 (27% greater than CSC) but not in 2021, and MRM tree height was greater in 2021 (7% greater than CSC). Mortality was significantly greater with MRM in both 2020 and 2021(in 2021: 32% vs. 17% under CSC), and MRM trees had more chlorotic leaves. Because of staff limitations, plant debris and soil were not routinely cleared from MRM, thus diminishing any benefit from the reflective surface. Better maintenance might have resulted in more sustained evidence of MRM growth benefits. With the current resource availability, the MRM system does not appear to accelerate the assessment of hybrid seedling trees. [ABSTRACT FROM AUTHOR]

Assessments of the resistance of citrus germplasm to huanglongbing (HLB) can be expedited by inoculating plants under laboratory or greenhouse settings with the HLB bacterium, Candidatus Liberibacter asiaticus (CLas). Consistent rapid screening is critical to efficiently assess disease resistance among plant materials; however, a number of factors may govern the efficacy of such inoculations. Despite the rapidity at which HLB can spread in a grove, it often takes 8 to 10 months for high levels of CLas and HLB symptoms to develop even in highly susceptible sweet orange. Therefore, two experiments were conducted to assess factors that might influence efficiency in screening for HLB resistance. In one experiment, three test citrus genotypes ('Kuharske', previously shown to be HLB resistant; rough lemon, previously shown to be HLB tolerant; and 'Valencia', HLB susceptible) were bud grafted using CLas-infected buds from four different source genotypes. All bud source genotypes had similar levels of CLas titer, but citron, rough lemon, and Volkamer lemon were hypothesized to be better bud inoculum sources as they are more tolerant of HLB than 'Valencia'. Among the three test genotypes over all sources of infected buds, inoculations of 'Kuharske' resulted in lower CLas titers and fewer HLB symptoms than inoculations of rough lemon or 'Valencia'. Inoculations of rough lemon resulted in higher CLas titers and more pronounced HLB symptoms when it was inoculated using infected buds from rough lemon or 'Valencia'. Grafting 'Valencia' with infected buds from Volkamer lemon resulted in less disease than when 'Valencia' was grafted with infected citron, rough lemon, or 'Valencia' buds. Overall, these results suggest that the source of CLas-infected buds used to graftinoculate some genotypes will influence disease development. Trunk cross-sectional area increase for the year following infection was 33 higher in 'Kuharske' and rough lemon, compared with 'Valencia'. 'Kuharske' had very low levels of CLas (30 CLas/µg DNA), whereas 'Valencia' (43,000 CLas/µg DNA) and rough lemon (6700 CLas/ µg DNA) had relatively high levels. As an alternative to graft-inoculating plants with CLas-infected buds, plants can be subjected to infestations of CLas-infected Asian citrus psyllid (ACP) as occurs naturally. Of interest is if transmission rates of CLas and the development of HLB in a genotype are greater when the ACP have been feeding on the same host genotype. An experiment was therefore conducted to assess transmission of CLas by ACP reared on CLas-infected rough lemon to five different genotypes ('Carrizo', 'Flame' grapefruit, rough lemon, 'Temple', and 'Valencia'). These assessments were made using a detached leaf assay recognized as a faster method of gauging transmission rates of CLas than using whole plants. Higher percentages of ACP died when they were transferred from infected rough lemon to healthy 'Carrizo', and lower percentages died when they were transferred to rough lemon or 'Flame'. However, CLas transmission by infected ACP occurred to at least some leaves of each genotype in each of the five different assays, with 70% or more leaves of each genotype becoming infected in at least one assay. Over all assays, there was relatively little variation among genotypes in the percentage of leaves becoming CLas infected and in the titer of CLas developing in infected leaves. However, there were relatively large differences in transmission rates among individual assays unrelated to differences among test genotypes. Because of the rapidity of the detached leaf assay, efforts are merited to improve consistency of this inoculation method. [ABSTRACT FROM AUTHOR]

A test population consisting of progenies of 92 seed-source genotypes (hereafter called "parent genotypes") of Citrus and Citrus relatives in the field in east-central Florida was assessed after natural freeze events in the winters of 2010 and 2011. Eight seedlings per parent genotype were planted in a randomized complete block design; however, as a result of mortality, the number of plants assessed in some genotype groups was reduced at some or all sampling dates. The citrus diseases huanglongbing and citrus canker were endemic in the planting and may have influenced tree response to cold temperatures. Unusually low temperatures (near -4 °C each winter) for east-central Florida were experienced during the trial period. Defoliation and dieback were significantly greater in the winter of 2011 than in the winter of 2010. The winter in 2011 was preceded by a period of extraordinarily low temperatures in mid-December with no period of cool temperatures to allow trees to acclimate. In 2010 the average defoliation was 53% ± 28% and less than 13% of the trees exhibited any noticeable dieback, whereas in 2011, the average defoliation and dieback were 93% ± 17% and 51% ± 35%, respectively. Within the genus Citrus, several progenies were identified that had 16% to 24% dieback in 2011 and these were from parent genotypes C. reticulata (CRC 2590) (23%), C. sinensis (CRC 3858) (24%), C. maxima (CRC 3945) (16%), C. hassaku (CRC 3907 and 3942) (16% and 17%), C. aurantium (CRC 628 and 2717) (18% and 7%), C. taiwanica (CRC 2588) (21%), and C. neo-aurantium (C. obovoidea + C. unshiu graft chimera) (CRC 3816) (23%). Within other genera in the Aurantiodeae, Poncirus trifoliata (CRCs 301,3957,3549, and 4007), Severinia buxifoiia (CRC 1497), Bergera koenigii (CRC 3165), and Glycosmis pentaphylla (CRC 3285) had the least amount of dieback, all at less than 23%. The two species within the Toddalioideae subfamily of the Rutaceae (Casimiroa edulis and Zanthoxylum ailanthoides) had among the least amount of dieback (1% and 8%, respectively). When considered by groups, the Citrons and Australian natives had the greatest amount of dieback in 2011, 68% and 65%, respectively. The trifoliates (Poncirus and hybrids) had the least dieback, ranging from 4% to 40%. The information from this study may be useful in germplasm enhancement and Citrus breeding targeting greater cold tolerance. [ABSTRACT FROM AUTHOR]

Transgenic solutions are being widely explored to develop huanglongbing (HLB) resistance in citrus. A critical component of a transgenic construct is the promoter, which determines tissue specificity and level of target gene expression. This study compares the characteristics of five promoters regulating the beta-glucuronidase ( GUS) reporter gene in the trifoliate hybrid rootstock US-802. Two of the selected promoters direct high levels of constitutive transgene expression in other dicotyledonous plants: 2X35S, the tandem-repeat promoter of the cauliflower mosaic virus 35S gene and bul409S, a truncation of the potato polyubiquitin promoter. Because Candidatus Liberibacter, the Gram-negative bacterium associated with HLB, infects only the phloem tissue, it may be advantageous to limit transgene expression to the vascular tissue and reduce expression in the fruit. Thus, we also tested three promoters that demonstrate phloem specificity when transformed and expressed in other plants: WDV, from wheat dwarf geminivirus; AtSUC2, the sucrose-H+ symporter gene promoter from Arabidopsis; and CsSUS, the sucrose synthase promoter from citrus. Histochemical staining for GUS activity was observed throughout leaf and stem tissues for the constitutive promoters, while the three phloem-specific promoters largely showed the expected tissue-specific staining. Expression of GUS in some individual transformants with promoters CsSUS and WDV appeared leaky, with some laminar tissue staining. Relative quantification of qRT-PCR data revealed a wide range of mRNA abundance from transgenics with each of the five promoters. Fluorometry also revealed that GUS activity differed depending on the promoter used, but mRNA levels and enzyme activity were not highly correlated. [ABSTRACT FROM AUTHOR]

In spring 1999, a commercial NAA (1-naphthaleneacetic acid) preparation for trunk sprout inhibition was compared with a corrugated plastic trunk wrap, aluminum foil wrap, bimonthly hand removal of sprouts, use of NAA preparation plus bimonthly hand removal when sprouts appeared, and a nontreated control. Three recently planted groves on three different rootstocks [Midsweet' orange (Citrus sinensis)] un Swingle citrumelo (Citrus paradisi x Poncirus trifoliata), 'Valencia' orange on Volkamer lemon (Volk, Citrus limon), and 'Minneola' tangelo (Citrus paradisi x C. reticulata) on Smooth Flat Seville (SFS, Citrus hybrid) received each of the treatments in a randomized complete block experimental design with trees blocked by initial height and circumference. Every 2 months, sprouts were counted on each tree and removed from the hand removal treatments. After 1 year, all sprouts were removed and counted and height and circumference of trees was determined. Across all experiments, 82% to 100% of nontreated trees produced trunk sprouts and all sprout control methods significantly reduced sprouts per tree. NAA treatments were never significantly less effective at sprout suppression than the wraps at the P = 0.05 level, although in two experiments, wraps were more effective than NAA at P = 0.10. Time of sprout appearance varied between the three experimental blocks. Plastic and foil trunk wraps enhanced development of trunk circumference compared with nontreated controls in 'Midsweet'/Swingle and 'Valencia'/ Volk. Greater trunk circumference resulted from use of wraps versus NAA in all three experiments, which appeared unrelated to differential sprout suppression. In these experiments, it appears that either wraps enhanced tree development beyond the suppression of sprouts or NAA influence on tree metabolism somewhat reduced trunk growth. The economics of the sprout suppression methods are also discussed. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is the most devastating disease for citrus worldwide. Candidatus Liberibacter asiaticus (CLas), vectored by Asian citrus psyllid (ACP, Diaphorina citri Kuwayama), is the most common pathogen causing the disease. Commercial citrus varieties are highly susceptible to HLB, whereas trifoliate orange (Poncirus trifoliata) is considered highly tolerant to HLB. An F1 segregating population and their parent trifoliate orange and sweet orange, which had been exposed to intense HLB pressure for three years, was evaluated for disease symptoms, ACP colonization, CLas titer and tree vigor repeatedly for two to three years. Trifoliate orange and sweet orange showed significant differences for most of the phenotypic traits, and the F1 population exhibited a large variation. A high-density SNP-based genetic map with 1 402 markers was constructed for trifoliate orange, which exhibited high synteny and high coverage of its reference genome. A total of 26 quantitative trait locus (QTLs) were identified in four linkage groups LG-t6, LG-t7, LG-t8 and LG-t9, of which four QTL clusters exhibit a clear co-localization of QTLs associated with different traits. Through genome-wide analysis of gene expression in response to CLas infection in 'Flying Dragon' and 'Larger-Flower DPI-50-7' trifoliate orange, 85 differentially expressed genes were found located within the QTL clusters. Among them, seven genes were classified as defense or immunity protein which exhibited the highest transcriptional change after CLas infection. Our results indicate a quantitative genetic nature of HLB tolerance and identified candidate genes that should be valuable for searching for genetic solutions to HLB through breeding or genetic engineering. [ABSTRACT FROM AUTHOR]

Citrus Huanglongbing (HLB) associated with ‘Candidatus Liberibacter asiaticus’ (Las) and citrus canker disease incited by Xanthomonas citri are the most devastating citrus diseases worldwide. To control citrus HLB and canker disease, we previously screened over forty antimicrobial peptides (AMPs) in vitro for their potential application in genetic engineering. D2A21 was one of the most active AMPs against X. citri, Agrobacterium tumefaciens and Sinorhizobium meliloti with low hemolysis activity. Therefore, we conducted this work to assess transgenic expression of D2A21 peptide to achieve citrus resistant to canker and HLB. We generated a construct expressing D2A21 and initially transformed tobacco as a model plant. Transgenic tobacco expressing D2A21 was obtained by Agrobacterium-mediated transformation. Successful transformation and D2A21 expression was confirmed by molecular analysis. We evaluated disease development incited by Pseudomonas syringae pv. tabaci in transgenic tobacco. Transgenic tobacco plants expressing D2A21 showed remarkable disease resistance compared to control plants. Therefore, we performed citrus transformations with the same construct and obtained transgenic Carrizo citrange. Gene integration and gene expression in transgenic plants were determined by PCR and RT-qPCR. Transgenic Carrizo expressing D2A21 showed significant canker resistance while the control plants showed clear canker symptoms following both leaf infiltration and spray inoculation with X. citri 3213. Transgenic Carrizo plants were challenged for HLB evaluation by grafting with Las infected rough lemon buds. Las titer was determined by qPCR in the leaves and roots of transgenic and control plants. However, our results showed that transgenic plants expressing D2A21 did not significantly reduce Las titer compared to control plants. We demonstrated that transgenic expression of D2A21 conferred resistance to diseases incited by P. syringae pv. tabaci and X. citri but not Las. Our results underscore the difficulty in controlling HLB compared to other bacterial diseases. [ABSTRACT FROM AUTHOR]

Hybrids of Poncirus trifoliata L. Raf. with Citrus have shown degrees of tolerance to the deadly citrus greening disease, hence prompting interest as potential commercial varieties. Although P. trifoliata is known to produce fruit that is inedible, fruit from many advanced hybrid trees have not been evaluated for their quality potential. The sensory quality of selected Citrus hybrids with varying degrees of P. trifoliata in their pedigrees is reported herein. Four Citrus × P. trifoliata hybrids developed through the USDA Citrus scion breeding program—1‐76‐100, 1‐77‐105, 5‐18‐24, and 5‐18‐31—had acceptable eating quality and sweet and sour taste, with mandarin, orange, fruity–noncitrus, and floral flavors. On the other hand, hybrids with higher proportion of P. trifoliata in their pedigrees, US 119 and 6‐23‐20, produced a juice characterized by green, cooked, bitter, and Poncirus‐like flavor and aftertaste. Partial least square regressions revealed that the Poncirus‐like off‐flavor is likely due to a combination of higher than typical amounts of sesquiterpene hydrocarbons (woody/green odor), monoterpenes (citrus/pine), and terpene esters (floral) and a lack of aldehydes with typical citrus odor (octanal, nonanal, and decanal). Sweetness and sourness were mostly explained by high sugars and acids, respectively. Further, carvones and linalool contributed to sweetness in the samples from early and late seasons, respectively. In addition to highlighting chemical contributors to sensory descriptors in Citrus × P. trifoliata hybrids, this study provides useful information on sensory quality for future citrus breeding efforts. Practical Application: The relationships between the sensory quality and secondary metabolites of Citrus × P. trifoliata hybrids described in this study help identify disease‐resistant Citrus scion hybrids with acceptable flavor and help mobilize this resistance in future breeding efforts. It also shows potential of such hybrids to be commercialized. [ABSTRACT FROM AUTHOR]

The level of anthocyanins in plants vary widely among cultivars, developmental stages and environmental stimuli. Previous studies have reported that the expression of various MYBs regulate anthocyanin pigmentation during growth and development. Here we examine the activity of 3 novel R2R3-MYB transcription factor (TF) genes,PamMybA.1, PamMybA.3andPamMybA.5fromPrunus americana. The anthocyanin accumulation patterns mediated byCaMV double35Spromoter (db35Sp) controlled expression of the TFs in transgenic tobacco were compared with citrus-MoroMybA, Arabidopsis-AtMybA1and grapevine-VvMybA1transgenics during their entire growth cycles. Thedb35Sp-PamMybA.1anddb35Sp-PamMybA.5 constructs induced high levels of anthocyanin accumulation in both transformed tobacco calli and the regenerated plants. The red/purple color pigmentation induced in thePamMybA.1andPamMybA.5 lines was not uniformly distributed, but appeared as patches in the leaves, whereas the flowers showed intense uniform pigmentation similar to theVvMybA1expressing lines.MoroMybAandAtMybA1showed more uniform pink coloration in both vegetative and reproductive tissues. Plant morphology, anthocyanin content, seed viability, and transgene inheritance were examined for thePamMybA.5transgenic plants and compared with the controls. We conclude that these TFs alone are sufficient for activating anthocyanin production in plants and may be used as visible reporter genes for plant transformation. Evaluating these TFs in a heterologous crop species such as citrus further validated that these genes can be useful for the metabolic engineering of anthocyanin production and cultivar enhancement. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is a serious and devastating disease of citrus caused by Candidatus Liberibacter spp. and vectored by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The disease has the potential to greatly limit the production of citrus in Florida and other citrus-growing regions worldwide. Current control of D. citri and HLB is inadequate, but the identification and incorporation of D. citri resistance traits from uncultivated Citrus spp. and Citrus relatives is seen as a potential disease management strategy. In this study, seedlings of 87 Rutaceae seed-source genotypes, primarily in the orange subfamily Aurantioideae, were assessed in the field for their propensity in a free-choice situation for infestations of natural south Florida populations of D. citri. The majority of test populations surveyed hosted all three life stages of D. citri; however, there were significant differences among the test populations in the mean ranks for 19. citri eggs (F = 3.13, df = 86, P < 0.0001), nymphs (F = 9.01, df 86, P < 0.0001), and adults (F = 4.21, df 86, P < 0.0001). The only sample test population that was completely avoided by all life stages of D. citri was seedlings of Casimiroa edulis Llave et Lex, commonly known as white sapote, which was one of the few plants included in the study belonging to the Rutaceae subfamily Toddalioideae. Although not completely avoided, very low levels of D. citri were found on two surveyed test populations of Poncirus trifoliata (L.) Raf, seedlings of 'Simmon's trifoliate' and 'Little-Leaf'. Poncirus trifoliata, the trifoliate orange, readily forms hybrids with Citrus spp., is commonly incorporated into rootstock varieties, and has been used in breeding-advanced scion material. The identification of partial resistance in P. trifoliata to D. citri could prove useful in future citrus breeding efforts aimed at reducing the incidence and spread of HLB. Determining if there is true resistance to D. citri in this and other germplasm sources with a low propensity for infestations in free-choice situations will require no-choice experiments. [ABSTRACT FROM AUTHOR]

Genetic traits in Poncirus trifoliata (Rutaceae) are known to confer resistance to colonization by the Asian citrus psyllid, but field surveys indicated these traits were not expressed in any of 24 simple hybrids between P. trifoliata and sweet orange. No resistance to colonization by the psyllid was observed in 14 other types of P. trifoliata hybrids. Three citrumelo hybrids appeared to be resistant in 1 survey, but in another survey, none of 6 citrumelos exhibited resistance. Interestingly, colonization was somewhat reduced on a hybrid with P. trifoliata in the pedigree of both parents, suggesting the possibility that traits suppressing oviposition may be recovered through similar crosses. [ABSTRACT FROM AUTHOR]

• Citrus x Poncirus hybrids have poor eating quality due to the Poncirus parent • In this study, three such hybrids had composition analysis similar to citrus fruit • Two hybrids had volatile composition closer to that of Poncirus trifoliata fruit • One hybrid had a flavonoid aglycone profile similar to that of pure Poncirus • A pattern of inheritance of Poncirus chemical traits could be seen Citrus breeders have made numerous hybridizations that included Poncirus trifoliata L.Raf. (P. trifoliata), a relative of the genus Citrus , in their pedigree to produce hybrids that are cold- and disease-resistant. Initial hybrids of Citrus × P. trifoliata typically produce fruit that have unacceptable flavor, but more advanced hybrids with greater proportions of Citrus in their pedigrees have various degrees of off-flavor and taste. This study reports the volatile constituents, as well as nonvolatile limonoids and flavonoids, in the juice of six Citrus hybrids containing P. trifoliata in their pedigrees, compared to two hybrids derived solely from Citrus. Secondary metabolite profiles reflected the complex genetic backgrounds of those hybrids. Hybrids 6_23−20 and US 119 had juice composition most similar to that of P. trifoliata , with more volatiles, and in particular, more esters and sesquiterpenes with higher amounts in total. Furthermore, hesperidin concentration in US 119 was low (10 μg g−1) in comparison with other hybrids (125−326 μg g−1) while concentrations of narirutin and isosakuranetin-7-O-rutinoside were unusually high (135−211 μg g−1 and 304−182 μg g−1, respectively) in comparison with the other hybrids (5−60 μg g−1 and 10−75 μg g−1, respectively), which is in agreement with the composition of pure P. trifoliata. Juice of P. trifoliata derived hybrids 5−18-24, 5−18-31, 1−76-100, and 1−77-105 presented secondary metabolite compositions closer to those of pure Citrus hybrids. Based on volatiles, limonoids, and flavonoid profiles, we conclude that it is possible to generate advanced Citrus hybrids including P. trifoliata in their pedigrees that approach the quality of common Citrus types. [ABSTRACT FROM AUTHOR]

Background: Most bacteria are not culturable, but can be identified through molecular methods such as metagenomics studies. Due to specific metabolic requirements and symbiotic relationships, these bacteria cannot survive on typical laboratory media. Many economically and medically important bacteria are unculturable; including phloem-limited plant pathogens like Candidatus Liberibacter asiaticus (CLas). CLas is the most impactful pathogen on citrus production, is vectored by the Asian citrus psyllid (ACP, Diaphorina citri), and lacks an effective treatment or resistant cultivars. Research into CLas pathogenicity and therapy has been hindered by the lack of persistent pure cultures. Work to date has been mostly limited to in planta studies that are time and resource intensive. Results: We developed and optimized an in vitro protocol to quickly test the effectiveness of potential therapeutic agents against CLas. The assay uses intact bacterial cells contained in homogenized tissue from CLas-infected ACP and a propidium monoazide (PMA) assay to measure antimicrobial activity. The applicability of PMA was evaluated; with the ability to differentiate between intact and disrupted CLas cells confirmed using multiple bactericidal treatments. We identified light activation conditions to prevent PCR interference and identified a suitable positive control for nearly complete CLas disruption (0.1% Triton-X 100). Isolation buffer components were optimized with 72 mM salt mixture, 1 mM phosphate buffer and 1% glycerol serving to minimize unwanted interactions with treatment and PMA chemistries and to maximize recovery of intact CLas cells. The mature protocol was used to compare a panel of peptides already under study for potential CLas targeting bactericidal activity and identify which were most effective. Conclusion: This psyllid homogenate assay allows for a quick assessment of potential CLas-disrupting peptides. Comparison within a uniform isolate largely eliminates experimental error arising from variation in CLas titer between and within individual host organisms. Use of an intact vs. disrupted assay permits direct assessment of potential therapeutic compounds without generating pure cultures or conducting extensive in planta or field studies. [ABSTRACT FROM AUTHOR]