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The global seed market is considered to be an important industry with a total value of $10,543 million US dollars in 2012. Because plant pathogens such as bacteria and viruses cause a significant economic loss to both producers and exporters, the seed export industry urgently requires rapid, sensitive, and inexpensive testing for the pathogens to prevent disease spreading worldwide. This study developed an antibody array in a multiwell plate format to simultaneously detect four crucial plant pathogens, namely, a bacterial fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), Chilli veinal mottle virus (ChiVMV, potyvirus), Watermelon silver mottle virus (WSMoV, tospovirus serogroup IV), and Melon yellow spot virus (MYSV, tospovirus). The capture antibodies specific to the pathogens were immobilized on each well at preassigned positions by an automatic microarrayer. The antibodies on the arrays specifically captured the corresponding pathogens present in the sample extracts. The presence of pathogens bound on the capture antibodies was subsequently detected by a cocktail of fluorescently conjugated secondary antibodies. The limits of detection of the developed antibody array for the detection of Aac, ChiVMV, WSMoV, and MYSV were 5 × 10(5) CFU/mL, 30 ng/mL, 1000 ng/mL, and 160 ng/mL, respectively, which were very similar to those of the conventional ELISA method. The antibody array in a multiwell plate format accurately detected plant pathogens in single and multiple detections. Moreover, this format enables easy handling of the assay at a higher speed of operation.

Acidovorax citrulli causes bacterial fruit blotch of cucurbits, a serious economic threat to watermelon (Citrullus lanatus) and melon (Cucumis melo) production worldwide. Based on genetic and biochemical traits, A. citrulli strains have been divided into two distinct groups: group I strains have been mainly isolated from various non-watermelon hosts, while group II strains have been generally isolated from and are highly virulent on watermelon. The pathogen depends on a functional type III secretion system for pathogenicity. Annotation of the genome of the group II strain AAC00-1 revealed 11 genes encoding putative type III secreted (T3S) effectors. Due to the crucial role of type III secretion for A. citrulli pathogenicity, we hypothesized that group I and II strains differ in their T3S effector repertoire. Comparative analysis of the 11 effector genes from a collection of 22 A. citrulli strains confirmed this hypothesis. Moreover, this analysis led to the identification of a third A. citrulli group, which was supported by DNA:DNA hybridization, DNA fingerprinting, multilocus sequence analysis of conserved genes, and virulence assays. The effector genes assessed in this study are homologous to effectors from other plant-pathogenic bacteria, mainly belonging to Xanthomonas spp. and Ralstonia solanacearum. Analyses of the effective number of codons and gas chromatography content of effector genes relative to a representative set of housekeeping genes support the idea that these effector genes were acquired by lateral gene transfer. Further investigation is required to identify new T3S effectors of A. citrulli and to determine their contribution to virulence and host preferential association.

Melon and watermelon bacterial fruit blotch, incited by Acidovorax citrulli, is limited to some areas in Brazil but causes important losses, mainly in melon-producing regions. Although genetic diversity has been observed among strains belonging to the species, they are considered a homogeneous group based on the fact that they show only slight physiological or nutritional differences. The objective of this study was to compare Brazilian strains from melon and watermelon by means of biochemical, pathogenicity, serological and molecular assays. Fifteen biochemical tests, cross inoculation between strains and hosts, ELISA and repetitive sequence analysis (rep-PCR) with the primers REP, ERIC and BOX were conducted. No differences were revealed by nutritional characterization or serology, but cross inoculation showed different pathogenicity groups, which could explain high aggressiveness of the bacteria to melon crops in some regions. Molecular analysis by BOX-PCR clustered strains according to their geographical origin, while ERIC- and REP-PCR, analyzed together, indicated genetic diversity, but without geographical or host origin relationships. One test that could be used to verify the pathogenicity of strains by inoculating detached leaf petioles, showing results in 36 h, is proposed here.

To assess the diversity of Acidovorax avenae subsp. citrulli, 121 strains from watermelon, cantaloupe, and pumpkin were compared using pulse field gel electrophoresis of SpeI-digested DNA and gas chromatographic analysis of fatty acid methyl esters. Twenty-nine unique DNA fragments resulted from DNA digestion, and 14 distinct haplotypes were observed. Based on cluster analysis, two subgroups, I and II, were recognized, which accounted for 84.8% (eight haplotypes) and 15.2% (six haplotypes) of the strains, respectively. Results of cellular fatty acid analysis varied quantitatively and qualitatively for the A. avenae subsp. citrulli strains and supported the existence of the two subgroups. Group I includes strains from cantaloupe and pumpkin as well as the ATCC type strain, which was first described in the United States in 1978, whereas group II represents the typical watermelon fruit blotch-causing strains that appeared in the mainland United States in 1989. Knowledge of the two A. avenae subsp. citrulli groups may be useful in screening for watermelon fruit blotch resistance.

Watermelon (Citrullus lanatus [Thunb.] Matsum & Nakai) fruit are affected by a number of preharvest disorders that may limit their marketability and thereby restrict economic returns to growers. Pathogenic diseases discussed include bacterial rind necrosis (Erwinia sp.), bacterial fruit blotch [Acidovorax avenae subsp. citrulli (Schaad et al.) Willems et al.], anthracnose [Colletotrichum orbiculare (Berk & Mont.) Arx. syn. C. legenarium (Pass.) Ellis & Halst], gummy stem blight/black rot [Didymella bryoniae (Auersw.) Rehm], and phytophthora fruit rot (Phytophthora capsici Leonian). One insect-mediated disorder, rindworm damage is discussed. Physiological disorders considered are blossom-end rot, bottleneck, and sunburn. Additionally, cross stitch, greasy spot, and target cluster, disorders of unknown origin are discussed. Each defect is shown in color for easy identification.

Acidovorax citrulli causes bacterial fruit blotch (BFB) of cucurbits, a disease that threatens the cucurbit industry worldwide. Despite the economic importance of BFB, little is known about pathogenicity and fitness strategies of the bacterium. We have observed the phenomenon of phenotypic variation in A. citrulli. Here we report the characterization of phenotypic variants (PVs) of two strains, M6 and 7a1, isolated from melon and watermelon, respectively. Phenotypic variation was observed following growth in rich medium, as well as upon isolation of bacteria from inoculated plants or exposure to several stresses, including heat, salt and acidic conditions. When grown on nutrient agar, all PV colonies possessed a translucent appearance, in contrast to parental strain colonies that were opaque. After 72 h, PV colonies were bigger than parental colonies, and had a fuzzy appearance relative to parental strain colonies that are relatively smooth. A. citrulli colonies are generally surrounded by haloes detectable by the naked eye. These haloes are formed by type IV pilus (T4P)-mediated twitching motility that occurs at the edge of the colony. No twitching haloes could be detected around colonies of both M6 and 7a1 PVs, and microscopy observations confirmed that indeed the PVs did not perform twitching motility. In agreement with these results, transmission electron microscopy revealed that M6 and 7a1 PVs do not produce T4P under tested conditions. PVs also differed from their parental strain in swimming motility and biofilm formation, and interestingly, all assessed variants were less virulent than their corresponding parental strains in seed transmission assays. Slight alterations could be detected in some DNA fingerprinting profiles of 7a1 variants relative to the parental strain, while no differences at all could be seen among M6 variants and parental strain, suggesting that, at least in the latter, phenotypic variation is mediated by slight genetic and/or epigenetic alterations.

Dutta, B., Avci, U., Hahn, M. G., and Walcott, R. R. 2012. Location of Acidovorax citrulli in infested watermelon seeds is influenced by the pathway of bacterial invasion. Phytopathology 102:461-468. Watermelon seeds can become infested by Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits via penetration of the ovary pericarp or by invasion of the pistil. This study investigated the effect of these invasion pathways on A. citrulli localization in seeds. Seed samples (n = 20 or 50 seeds/lot) from pistil- and pericarp-inoculated lots were dissected into testa, perisperm-endosperm (PE) layer, and embryo tissues and tested for A. citrulli by species-specific polymerase chain reaction (PCR) and by plating on semiselective media. Less than 8% of the testa samples were A. citrulli-positive regardless of the method of seed inoculation. Additionally, the difference in percentages of contaminated testae between the two seed lot types was not significant (P = 0.64). The percentage of A. citrulli-positive PE layer samples as determined by real-time PCR assay was significantly greater for seeds from pistilinoculated lots (97%) than for seeds from pericarp-inoculated lots (80.3%). The mean percentage of A. citrulli-positive embryo samples was significantly greater for seeds from pistil-inoculated lots (94%) than for seeds from pericarp-inoculated lots (≈8.8%) (P = 0.0001). Removal of PE layers and testae resulted in a significant reduction in BFB seed-toseedling transmission percentage for seeds from pericarp-inoculated lots (14.8%) relative to those from pistil-inoculated lots (72%). Additionally, using immunofluorescence microscopy, A. citrulli cells were observed in the PE layers and the cotyledons of pistil-inoculated seeds but only in the PE layers of pericarp-inoculated seeds. These results suggest that pericarp invasion results in superficial contamination of the testae and PE layers while pistil invasion results in the deposition of A. citrulli in seed embryos.

Prevention of the introduction of bacterial fruit blotch of watermelon, caused by Acidovorax avenae subsp. citrulli, into the transplant house or field is the most effective control strategy. Watermelon seedlots currently are screened for A. avenae subsp. citrulli, but other cucurbits, often grown in the same transplant house or field, generally are not as carefully monitored. In 1997 and 1999 field tests, cultivars of watermelon, muskmelon, honeydew melons, acorn squash, butternut squash, yellow squash, zucchini squash, cucumber, and pumpkin were evaluated for foliar and fruit susceptibility to bacterial fruit blotch and for seed transmission of A. avenae subsp. citrulli. The bacterium was introduced into the field on infected watermelon transplants or by misting a bacterial suspension onto fruit of the cucurbits. Foliar and fruit symptoms were more extensive in the watermelon, muskmelon, and honeydew melons than in the other cucurbits. In greenhouse grow-out assays, seed transmission of A. avenae subsp. citrulli was detected in every cucurbit in at least one of the two seasons, even though there were no fruit symptoms in some of them. Thus, any cucurbit crop plant should be considered a potential source for the introduction of A. avenae subsp. citrulli into the transplant house or field.

It is well known that bacterial fruit blotch (BFB) of watermelon caused by Acidovorax avenae subsp. citrulli threatens watermelon production. There is no pathogen enrichment or concentrated steps before detection in conventional PCR-based diagnostic technique, which consequently leads to the lower detection sensitivity. In the present study, two different bacterial suspensions were prepared, and four pathogen enrichment protocols including Biological Preparation (BP), Membrane Filtration (MF), Immune-Magnetic Separation (IMS) and Immune-Capture Preparation (ICP) were employed to prepare the PCR template for the pathogen detection. Double Antibody Sandwich Enzyme Linked Immunosorbent Assay (DAS-ELISA) was also conducted as a parallel comparison. The results showed that IC-PCR was the optimum method through comparing the detection limits, time and expenditure among those methods. The detection limit of IC-PCR for both bacterial suspension and seeds suspension can reach 102 CFU/ml, which was 10 times lower than that of IMS-PCR when the seed suspension was used as template. In addition, the results of IC-PCR showed higher degree of precision than those of IMS-PCR. The time and expenditure of IC-PCR were comparable with other methods. The present study demonstrated that the procedures of immune-capture PCR is a sensitive, specific, rapid, reproducible, and economical method to detect A. avenae subsp. citrulli in watermelon seeds. Key words: Bacterial Fruit Blotch, watermelon seed, pathogen enrichment, PCR.

Acidovorax citrulli causes seedling blight and bacterial fruit blotch of cucurbits. Previous reports demonstrated the contribution of type IV pili (T4P) to A. citrulli virulence and to systemic infection of melon seedlings. Microfluidic flow-chamber assays demonstrated the involvement of T4P in surface adhesion and biofilm formation, whereas polar flagella did not appear to contribute to either of these features. On the other hand, a transposon mutant impaired in the biosynthesis of polar flagella was identified in screens for reduced virulence of an A. citrulli mutant library. Further characterization of polar flagellum mutants confirmed that A. citrulli requires a polar flagellum for full virulence on melon plants. Foliage and stem inoculation experiments revealed that polar flagella contribute to A. citrulli virulence and growth in planta at both pre- and post-host-tissue penetration. Interestingly, light microscope observations revealed that almost all A. citrulli wild-type cells extracted from the xylem sap of stem-inoculated melon seedlings remained motile, supporting the importance of this organelle in virulence and colonization of the host vascular system. We also report a negative effect of polar flagellum impairment on T4P-mediated twitching motility of A. citrulli and discuss a possible co-regulation of these two motility machineries in this bacterium.

The aim of this work was to investigate the ability of Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits, to colonize melon tissues. Under greenhouse conditions leaves and seeds were inoculated with a spontaneous mutant of A. citrulli (group I) resistant to rifampicin (Ac1Rif), and samples of hypocotyls, cotyledonary leaves, roots, leaves and stems were processed at three-day intervals. When the 1st pair of melon leaves had been inoculated, A. citrulli was only detected until the 10th pair of leaves (1.3 x 10³ CFU g-1 of leaf) and the consecutive stem (3.3 x 10³ CFU g-1 of stem) on the 30th day. However, after seed inoculation A. citrulli colonized the hypocotyl, roots, cotyledonary leaves, leaves and stems and was only detected until the 4th pair of leaves (1.62 x 10² CFU g-1 of leaf) and the consecutive stem (9.1 x 10¹ CFU g-1 of stem) on the 24th day post-inoculation. In conclusion, A. citrulli colonized different parts of the melon plant over time, depending on its initial location, leaves or seed. This confirms what has been observed in the field, that expanded leaves and stems are the main inoculum sources for melon blossoms and fruit, therefore providing scientific bases for developing more effective strategies for BFB management. Este trabalho investigou a habilidade de Acidovorax citrulli, agente causal da mancha-aquosa em cucurbitáceas, colonizar plantas de meloeiro. Em condições de casa de vegetação, folhas e sementes foram inoculadas com um mutante espontâneo de A. citrulli (grupo I) resistente a rifampicina (Ac1Rif) e a intervalos de três dias, amostras de hipocótilo, folhas cotiledonares, raízes, folhas ou ramos foram processadas. Após a inoculação no 1º par de folhas, A. citrulli foi detectada até o 10º par de folhas (1,3 x 10³ UFC g-1 de folha) e no ramo consecutivo (3,3 x 10³ UFC g-1 de ramo) aos 30 dias. A partir da inoculação das sementes A. citrulli colonizou efetivamente o hipocótilo, raízes, folhas cotiledonares, folhas e ramos, sendo a bactéria detectada até o 4º par de folhas (1,62 x 10² UFC g-1 de folha) e no ramo consecutivo (9,1 x 10¹ UFC g-1 e ramo) aos 24 dias após a inoculação. Pode-se concluir que A. citrulli colonizou diferentes partes do meloeiro ao longo do tempo, dependendo da localização do inóculo inicial, folhas ou sementes. Isto confirma o que tem sido observado nas condições de campo, ou seja, que folhas expandidas e ramos são fontes de inóculo para flores e frutos de melão, dando, portanto, suporte científico para o desenvolvimento de estratégias mais efetivas de manejo da mancha-aquosa do meloeiro.

DNA-rRNA hybridizations, DNA-DNA hybridizations, polyacrylamide gel electrophoresis of whole-cell proteins, and a numerical analysis of carbon assimilation tests were carried out to determine the relationships among the phylogenetically misnamed phytopathogenic taxa Pseudomonas avenae, Pseudomonas rubrilineans, "Pseudomonas setariae," Pseudomonas cattleyae, Pseudomonas pseudoalcaligenes subsp. citrulli, and Pseudomonas pseudoalcaligenes subsp. konjaci. These organisms are all members of the family Comamonadaceae, within which they constitute a separate rRNA branch. Only P. pseudoalcaligenes subsp. konjaci is situated on the lower part of this rRNA branch; all of the other taxa cluster very closely around the type strain of P. avenae. When they are compared phenotypically, all of the members of this rRNA branch can be differentiated from each other, and they are, as a group, most closely related to the genus Acidovorax. DNA-DNA hybridization experiments showed that these organisms constitute two genotypic groups. We propose that the generically misnamed phytopathogenic Pseudomonas species should be transferred to the genus Acidovorax as Acidovorax avenae and Acidovorax konjaci. Within Acidovorax avenae we distinguished the following three subspecies: Acidovorax avenae subsp. avenae, Acidovorax avenae subsp. cattleyae, and Acidovorax avenae subsp. citrulli. Emended descriptions of the new taxa are presented.

Acidovorax avenae subsp. citrulli is the causal agent of bacterial fruit blotch (BFB), a threatening disease of watermelon, melon, and other cucurbits. Despite the economic importance of BFB, relatively little is known about basic aspects of the pathogen's biology and the molecular basis of its interaction with host plants. To identify A. avenae subsp. citrulli genes associated with pathogenicity, we generated a transposon (Tn5) mutant library on the background of strain M6, a group I strain of A. avenae subsp. citrulli, and screened it for reduced virulence by seed-transmission assays with melon. Here, we report the identification of a Tn5 mutant with reduced virulence that is impaired in pilM, which encodes a protein involved in assembly of type IV pili (TFP). Further characterization of this mutant revealed that A. avenae subsp. citrulli requires TFP for twitching motility and wild-type levels of biofilm formation. Significant reductions in virulence and biofilm formation as well as abolishment of twitching were also observed in insertional mutants affected in other TFP genes. We also provide the first evidence that group I strains of A. avenae subsp. citrulli can colonize and move through host xylem vessels.

Bacterial fruit blotch (BFB), caused by Acidovorax avenae subsp. citrulli, is a seed-borne disease of cucurbits that spreads rapidly in the warm, humid environment of the transplant house, often resulting in high numbers of infected plants going into the field. The only control options for BFB once it gets into a transplant house are crop destruction or multiple applications of a copper-containing bactericide/fungicide. In this study, various treatments were compared with the standard foliar spray application of cupric hydroxide for BFB control under transplant house conditions. Peroxyacetic acid at 80 μg/ml and ionized copper at 1.0 and 1.5 μg/ml applied through the daily irrigation water were more effective than cupric hydroxide in reducing spread of A. avenae subsp. citrulli. Combining ionized copper or peroxyacetic acid in the irrigation water with a weekly foliar application of acibenzolar-S-methyl was most effective in reducing spread. The utilization of these transplant house treatments along with the elimination of all transplants with symptoms or near plants with symptoms should greatly reduce the chances of introducing BFB into fields on transplants. Accepted for publication 14 November 2008. Published 29 January 2009.

Bacterial fruit blotch (BFB) is the most economically important bacterial disease of cucurbits worldwide. The pathogen is seed transmitted and affects all stages of many cucurbit crops eventually causing destructive fruit rots. Like many phytobacterial diseases, the chemical options for BFB management are limited and primarily include copper based compounds. Additionally, the unavailability of commercial cucurbits cultivars with BFB resistance makes disease management difficult. Out of necessity, BFB management involves an integrated approach that seeks to exclude primary inoculum through the production of clean seeds by isolating seed production fields seed field inspection and certification, seed health testing, seedling inspection, and prophylactic copper-based management. Thus far, despite the efforts to exclude Acidovorax avenae subsp. citrulli, BFB outbreaks continue to occur sporadically worldwide. To develop a more effective integrated disease management strategy, a better understanding of BFB epidemiology and pathogenesis is needed in fruit and seed production environments. Additionally, understanding the role of blossoms in seed infection has revealed potential avenues for integrated disease management. This chapter will explore the current understanding of the biology and epidemiology of BFB and the integrated disease management strategies currently employed.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-16T14:20:21Z No. of bitstreams: 1 Aldenir de Oliveira.pdf: 717930 bytes, checksum: eb22d1cac453b579b74b66da5fd0bc67 (MD5) Made available in DSpace on 2017-02-16T14:20:21Z (GMT). No. of bitstreams: 1 Aldenir de Oliveira.pdf: 717930 bytes, checksum: eb22d1cac453b579b74b66da5fd0bc67 (MD5) Previous issue date: 2008-02-29 This dissertation aimed to study: colonization of Acidovorax avenae subsp. citrulli in melons after inoculation of the first pair of true leaves, seeds and hermaphrodite flowers; bacterial survival in fruit and leaf residues incorporated to the soil at 0, 5, 10 and 15 cm depth and in soils without the host plant, under the influence of different soil type, temperature (10, 15, 20, 25, 30 and 35ºC) and humidity (50 and 100% of field capacity). In all studies a mutant resistant to 100 ppm of rifampicin (Aac1Rif) was utilized. Bacterial colonization was detected until 30 days after inoculation in the 10th pair of true leaves, with populations of 3.1 log UFC g-1 of leaf. In the same period, the shoot segment between the 10th and 11th leaf pair showed population of 3.52 log UFC g-1 of shoot. After seed inoculation the pathogen colonized the hypocotyl, roots, cotyledonary leaves, true leaves and shoots, until reach undetectable levels at 27 days after inoculation. Flower colonization by the bacteria was not verified. Aac1Rif was found in fruit and leaf residues at 0, 5 and 10 cm during 21 days and at 15 cm during 14 days. Highest population relative extinction rates (TERP)were presented by fruits on soil surface [0.1464 log (UFC) day -1] and on leaves at 10cm [0.084 log (UFC) day -1]. Aac1Rif survived on seven soil types only during three days and the soil C showed the highest TERP [0.9062 log (UFC) day-1]. Higher concentrations of Na+ and silt as well as higher populations of actinomycetes and Trichoderma correlated to faster extinction of Aac1Rif populations in soil. Generally for all soils the lowers TERP were found at 10 or 15ºC and the higher, at 30 or 35ºC. There was no significant (P=0.05) interaction between soil and humidity, however the T test showed significant difference (P=0.05) between the TERP at 100% [0.6685 log (UFC) day -1] and 50% [0.504591 log (UFC) day -1] of field capacity. Independent of temperature and humidity, Aac1Rif also survived in soil only during three days. Esta dissertação teve como objetivos estudar: colonização de A. avenae subsp. citrulli em meloeiro a partir da inoculação no primeiro par de folhas verdadeiras, sementes e flores hermafroditas; sobrevivência da bactéria em restos de folhas e frutos incorporados ao solo a diferentes profundidades (0, 5, 10 e 15 cm) e em solos na ausência da planta hospedeira, sob a influência de diferentes tipos de solo (sete solos), temperaturas (10, 15, 20, 25, 30 e 35ºC) e umidades (50 e 100% da capacidade de campo). Para os estudos foi utilizado um mutante resistente a 100 ppm de rifampicina (Aac1Rif). Quando a bactéria foi inoculada nas folhas, a colonização foi detectada até os 30 dias, no 10º par de folhas verdadeiras, com população de 3,1 log UFC g-1 de folha. Nesse mesmo período, observou-se a colonização no segmento de ramo compreendido entre o 10º e 11º par de folhas com população de 3,52 log UFC g-1 de ramo. A partir de sementes, a bactéria colonizou o hipocótilo, raízes, folhas cotiledonares, folhas verdadeiras e ramos, até atingir níveis não detectáveis aos 27 dias após a inoculação. Não foi verificada colonização das flores pela bactéria. Aac1Rif foi encontrada em restosde frutos e folhas de meloeiro a 0, 5 e 10 cm durante 21 dias e a 15 cm por 14 dias. As maiores taxas de extinção relativa da população (TERP) ocorreram nos frutos na superfície do solo [0,1464 log (UFC) dia-1] e nas folhas a 10 cm [0,084 log (UFC) dia-1]. Aac1Rif sobreviveu nos sete tipos de solo apenas durante três dias e o solo C apresentou a maior TERP [0,9062 log (UFC) dia-1]. Maiores concentrações de Na+ e silte bem como maiores populações de actinomicetos e Trichoderma estiveram correlacionadas a mais rápida extinção da população de Aac1Rif no solo. Para a maioria dos solos, as menores TERP foram atingidas a 10 ou 15ºC e as maiores, a 30 ou 35ºC. Não houve interação significativa (P=0,05) entre solos e umidade, contudo o teste de T evidenciou diferença significativa (P=0,1) entre as TERP a 100% [0,6685 log (UFC) dia-1] e 50% [0,504591 log (UFC) dia-1] da capacidade de campo. Independente da temperatura e umidade, Aac1Rif também sobreviveu nos solos apenas por três dias.

Seed transmission of A. avenae subsp. citrulli is key in the dissemination of bacterial fruit blotch of cucurbits. This study reports its survival and seed transmission from watermelon and melon seed lots that were more than 30 yr old. The longest previously-reported survival of A. avenae subsp. citrulli was from 19-yr-old watermelon seed and the longest reported survival for any seedborne bacterial plant pathogen was 24 yr for Curtobacterium flaccumfaciens pv. flaccumfaciens from common bean seed. Accepted for publication 19 September 2008. Published 19 December 2008.

A mancha-aquosa, causada por Acidovorax avenae subsp. citrulli (Aac) causa grandes prejuízos à cultura do melão. O controle dessa doença foi estudado in vivo, com microbiolização de sementes de melão Amarelo infectadas, com líquidos fermentados de Bacillus subtilis R14, B. megaterium pv. cerealis RAB7, B. pumilus C116 e Bacillus sp. MEN2, com e sem células bacterianas. O mecanismo de ação dos isolados foi estudado in vitro pelo método de difusão em ágar e os compostos bioativos parcialmente caracterizados por testes de hemólise e atividade surfactante. Nos testes in vivo, não houve diferença significativa entre os tratamentos com e sem células, indicando que o controle ocorreu devido à presença de compostos bioativos produzidos durante as fermentações. Todos os tratamentos diferiram da testemunha sem diferir entre si (P=0,05%). B. megaterium pv. cerealis RAB7 proporcionou redução da incidência (89,1%) e do índice de doença (92,7%), elevou o período de incubação da mancha-aquosa de 9,8 para 11,9 dias e reduziu a AACPD de 3,36 para 0,17. In vitro, todos isolados apresentaram antibiose contra Aac e os compostos bioativos foram parcialmente caracterizados como lipopeptídeos. The bacterial fruit blotch, caused by the bacterium Acidovorax avenae subsp. citrulli (Aac), is responsible for great losses in melon production. The control of this disease was investigated in vivo by treating infected yellow melon seeds with fermented broths of B. subtilis R14, B. megaterium pv. cerealis RAB7, B. pumilus C116 and Bacillus sp. MEN2, with and without bacterial cells. The mechanism of action of the strains was studied in vitro by the agar diffusion technique. The bioactive compounds produced were partially characterized by hemolysis test and surfactant activity. Regarding the tests conducted in vivo there was no statistical difference between the treatments with and without bacterial cells, which indicated that the control was due to the presence of bioactive compounds produced during fermentations. All treatments were different from the control (P = 0.05%) but they did not differ among them. The B. megaterium pv. cerealis RAB7 reduced disease incidence (89.1%), disease index (92.7%), increased the incubation period from 9.8 to 11.9 days and reduced the area under disease progress curve from 3.36 to 0.17. In the in vitro tests, all strains showed antibiosis against Aac and the bioactive compounds were partially characterized as lipopeptides.

Acidovorax avenae subsp. citrulli (Pseudomonas pseudoalcaligenes subsp. citrulli), the causal agent of a watermelon seedling blight and fruit blotch (WFB), has emerged as a serious seedborne pathogen of watermelon and melons world-wide. The disease was first reported in 1965 in seedlings of several USDA Plant Introduction (PI) lines in Georgia. The pathogen was described 13 years later in diseased seedlings from two of the same PI seed sources in which Web and Goth had originally identified the disease. The disease remained known only in seedlings at the introduction station until Wall and Santos isolated a non-fluorescent pseudomonad from rotting fruit in Guam in 1987 and concluded it was the same organism. WFB was reported in Florida in 1989 and a year later emerged as a major economic disease of watermelon in the Southeast and Midwest USA. Since then, WFB has been reported in Turkey, Brazil, Australia, Japan, Korea, Thailand, and China infecting cantaloupe, melon, honeydew, pumpkin, and citron under field conditions. Because the original seedling strains were reported as giving a negative tobacco hypersensitive reaction and failed to rot fruit like the field disease strains, some have suggested that the originally described organism was not the same organism as that causing WFB. DNA fingerprinting of a collection of 121 strains using pulse field gel electrophoresis revealed considerable genetic diversity. All strains typed into two groups; over 80% were in Group I and the remaining, including the original type strain, in Group II. Control of the disease is based primarily on use of pathogen-free seed. Although attempts have been made to develop a routine seed assay to detect the organism in seeds, no reliable method is available. Contaminated seeds remain a problem for the seed industry.

In July 2013, a melon plant sample (Cucumis melo cv. Saski) with disease symptoms resembling bacterial fruit blotch (BFB), was collected from a 10-acre field located in Yolo County, California, and submitted to the Plant Pest Diagnostics Center of the CDFA. Melon leaves had small (5 to 10 mm in diameter), tan to dark reddish-brown, angular lesions surrounded by yellow halos, and larger V-shaped lesions that extended from the leaf margins to the midrib. Bacterial streaming was observed at 400× magnification. The bacterium isolated from a leaf tissue wet mount formed smooth, round, cream-colored, non-fluorescent colonies on Pseudomonas F agar, was gram-negative, rod-shaped, aerobic, and oxidase-positive. The strain grew at 41°C and produced a strong hypersensitive response on tobacco (Nicotiana tabacum) 24 h after tissue infiltration. Based on a positive immunoassay test for Acidovorax citrulli (Eurofins STA Lab, Inc., Longmont, CO) and positive real-time PCR assays using species-specific primer sets, BX-L1/BX-S-R2 (1) and ZUP2436/2437 (4), the strain was identified as A. citrulli. A 360-bp fragment of the 16S ribosomal RNA gene was amplified using conventional PCR with primers WFB1 and WFB2 (3). The fragment, GenBank Accession No. KJ531595, showed 100% identity with the corresponding regions of A. citrulli (CP000512) strain AAC00-1 by BLAST query. Pathogenicity tests were performed by injecting 0.5 to 1 ml suspensions of the bacteria (106 CFU/ml) under the rind of three mature honeydew fruit (Cucumis melo var. indorus), three watermelon fruit (Citrullus lanatus cv. Sugar Baby), and into the cotyledons of ten, 10-day-old watermelon seedlings (cv. Sugar Baby). The fruit and seedlings were incubated in plastic bags at 30°C and similar treatments with sterile water served as negative controls. After 4 days, the seedlings inoculated with the suspect strain exhibited dark brown necrotic lesions with yellow halos that later coalesced, causing the cotyledons to collapse. Seven days after inoculation, the honeydew fruit exhibited dry, rotten gray cavities (4 to 6 cm in diameter) in the pericarp tissue below the rind. In contrast, the watermelon fruit had completely collapsed in a watery rot after 7 days. No symptoms were observed on the negative control fruits and seedlings treated with water. The pathogen was re-isolated from the inoculated fruit and seedlings and confirmed as A. citrulli by species-specific PCR and immunoassay as described above. The melon seed lot used to plant the field in Yolo County, CA, also tested positive for A. citrulli using species-specific real-time PCR assays (1,4). DNA fingerprinting by pulse field gel electrophoresis of Spe I-digested whole cell genomic DNA showed that all of the California A. citrulli strains were members of subgroup II (haplotype C strain) (3). These haplotypes normally occur on watermelon. BFB is a seed-transmitted disease of cucurbits and a major concern for national and global seed trade. First observed in United States commercial watermelon fields in 1989, BFB can cause economic losses up to 90% for commercial watermelon, cantaloupe, and honeydew growers (1,2). While BFB routinely occurs in the southeastern United States, this is the first official record of the disease in California. References: (1) O. Bahar et al. Plant Pathol. 57:754, 2008. (2) R. Walcott et al. J. Phytopathol. 152:277, 2004. (3) R. Walcott et al. Plant Dis. 84:470, 2000. (4) B. Woudt et al. Phytopathology 99:S143, 2009.

Bacterial fruit blotch appeared in the United States in commercial watermelon fields with isolated severe outbreaks in Florida, South Carolina, and Indiana. Because the pathogen is seedborne, and a significant portion of commercial watermelon fields are planted with seedlings raised in greenhouses or other transplant production facilities, recognition of symptoms on seedlings is important. Posted 2 June 2000.

Typical bacterial fruit blotch (BFB) symptoms were observed on cvs. Crisby, Suzy, Top Gun, and Lady watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai.) fruits in southeastern Hungary (Medgyesegyháza) in July 2007 when the mean maximum daytime temperature was greater than 32°C. Approximately 20 to 30 ha of watermelons were observed to be affected by the disease. Source of the infection was not determined, however, grafted watermelon transplants that were grown in this area had been imported from Turkey where the pathogen is present (2). Disease symptoms started with irregularly shaped, water-soaked lesions on the surface of the fruits. The lesions enlarged and the epidermis became brown and cracked. BFB symptoms were not readily visible on mature foliage. Colonies of the BFB pathogen were creamy white on nutrient agar (Difco, Detroit, MI). Strains were gram negative, oxidase positive, and produced acid from glucose aerobically. A cell suspension (50 μl of ~1 × 107 CFU/ml) from a 24-h nutrient plate culture was infiltrated with a hypodermic syringe into the intercellular spaces of fully developed intact tobacco (Nicotiana tabacum L. cv. White Burley) leaves to determine the hypersensitive reaction (HR) (1). A typical HR developed 20 h after leaf infiltration. Bacterial pathogenicity was tested on surface-sterilized, mature fruits of different plant species by injecting cell suspensions into the fruit tissues as previously described (each fruit was injected in five places; the negative control (sterile water) as well). Fruits were incubated for 7 days at 25°C and then observed for symptom development. Necrosis was observed at each point of inoculation with the pathogen for watermelon and green pepper (Capsicum annuum L.). Necrosis was also observed for cucumber (Cucumis sativus L.), zucchini (Cucurbita pepo L. convar. giromontiina Greb.), squash (C. pepo L.), and patisson (C. pepo L. convar. patissoniana Greb.). Necrosis was not observed when the pathogen was inoculated onto fruit of melon (Cucumis melo L.), tomato (Lycopersicon esculentum Mill.), and eggplant (Solanum melongena L.). Additionally, symptoms were not observed at the points inoculated with sterile water (negative control) for any of the fruits tested. To identify the pathogen, PCR was used with Acidovorax avenae subsp. citrulli-specific primers WFB1/2 (4). The 16s rDNA region amplified with a general bacterial primer pair (63f forward and 1389r reverse) (3) was cloned into a pBSK+ vector (Stratagene, La Jolla, CA) and sequenced by M13 forward and reverse primers (GenBank Accession No. AM850114). On the basis of the symptoms, biochemical tests (API 20NE; Biomérieux, Marcy l'Etoile, France), fatty acid methyl ester analysis (74.5 to 83.6% similarity), and 16SrDNA sequence homology (100% sequence similarity with AAC00-1), the pathogen was identified as A. avenae subsp. citrulli. To our knowledge, this is the first report of BFB of watermelon in Hungary. References: (1) Z. Klement. Nature 199:299, 1963. (2) M. Mirik et al. Plant Dis. 6:829, 2006. (3) A. M. Osborn et al. Environ. Microbiol. 2:39, 2000. (4) R. R. Walcott and R. D. Gitaitis. Plant Dis. 84:470, 2000.

In August 2001, symptomatic mature fruit of watermelon (Citrullus lanatus) cvs. Crimson Sweet and Royal Sweet were collected from six fields (48 ha) in Iroquois, Kankakee, and Mason counties in Illinois. The fruit surface had dark green, water-soaked lesions, and the rind's epidermis was cracked. Sections of infected rind were examined with a light microscope, and bacterial streaming was observed. Rind tissue was disinfested by rubbing the fruit surface with a paper towel moistened with 70% ethanol. Pieces of rind from the margins of lesions were placed in a test tube containing sterile-distilled water. The tube was shaken, and the suspension was streaked on King's medium B agar in petri plates. The plates were incubated at 26°C for 3 days. A nonfluorescent, gramnegative, anaerobic, oxidase-positive, rod-shaped bacterium was isolated that produced creamy white colonies on nutrient agar. The bacterium was identified as Acidovorax avenae subsp. citrulli based on an enzyme-linked immunosorbent assay test specific for A. avenae subsp. citrulli (kit PSA 14,800; Agdia Inc., Elkhart, IN). The disease was diagnosed as bacterial fruit blotch (1,2). The occurrence of bacterial fruit blotch of watermelon in Illinois is important because (i) watermelon is widely grown in Illinois, (ii) the pathogen also infects melons and pumpkins, and (iii) Illinois grows approximately 1,000 ha of melons and 8,000 ha of pumpkins. References: (1) R. X. Latin. Bacterial fruit blotch. Pages 34-35 in: Compendium of Cucurbit Diseases. The American Phytopathological Society, St. Paul, MN,1996. (2) G. S. Somodi et al. Plant Dis. 75:1053, 1991.

Seedlings of 22 watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai] cultivars and two plant introductions were screened in the greenhouse for resistance to the fruit blotch bacterium. There were significant differences in disease severity among cultivars, but no cultivar was immune to the bacterium. In field tests, fruit of 18 commercial cultivars were inoculated individually or became infected naturally from diseased foliage. Cultivars with relatively resistant fruit included `Sugar Baby', `Jubilation', `Mirage', `Calsweet', `Crimson Sweet', `Royal Sweet', and `Sangria'. The more susceptible cultivars generally had a light-colored rind. Cultivar level of resistance to bacterial fruit blotch may not be sufficient under conditions conducive to severe disease development.

The watermelon fruit blotch organism (Acidovorax avenae subsp. citrulli) was confirmed as the cause of a bacterial disease of rockmelon seedlings in January 1996. Further outbreaks occurred in commercial nurseries during 1996–98. An associated field disease was not observed in rockmelon and honeydew crops until May 1998 when wet conditions led to severe leaf spotting and fruit infection in many crops in the Burdekin district of North Queensland. Isolates of A. avenae subsp. citrulli originating from these outbreaks were considerably more pathogenic to rockmelon plants than isolates originating from watermelon crops in South Queensland. They were also less pathogenic to the weed host Cucumis myriocarpus and could constitute new strains of the fruit blotch organism. Tests showed the disease was readily seed transmissible from naturally infected rockmelon and honeydew fruit for at least 3 months after seed extraction.

Citronmelon fruits (Citrullus lanatus var. citroides (Bailey) Mansf.) with lesions were collected from a cowpea field in Frio County, TX, in July 1997. The lesions were circular, necrotic, or water-soaked, approximately 3 mm in diameter, and did not extend into the flesh of the fruit. Nonfluorescent, gram-negative bacteria were consistently isolated from lesions. Six representative strains were identified as Acidovorax avenae subsp. citrulli (Aac), using Biolog GN MicroPlates and the MicroLog data base release 3.50 (0.533 to 0.813 similarity). Aac causes leaf and fruit lesions (bacterial fruit blotch, BFB) on watermelon (C. lanatus (Thunb.) Matsum. & Nakai). Strains were tested for pathogenicity on watermelon seedlings (cv. Royal Sweet) by daubing bacterial suspensions (approximately 108 CFU/ml) onto cotyledons of 1-week-old seedlings. Water soaking of cotyledons, followed by necrosis and seedling death, occurred within 5 days. These symptoms were indistinguishable from those caused by watermelon strains of Aac. Bacteria were reisolated from symptomatic seedlings. The source of the infection is not known. Watermelons had been grown in this field in 1996, but no BFB symptoms were observed. Citron fruit infected with Aac were found in nearby watermelon fields where BFB was present; the closest field was 50 m from the cowpea field. These observations suggest that citronmelon, a common weed in south Texas, has the potential to perpetuate Aac. This is the first documentation of a naturally occurring infection of citronmelon with Aac.

Honeydew fruits (Cucumis melo var. inodorus Jacq. ‘Morning Ice,’ ‘Honeybrew,’ and ‘Green Flesh’) with circular, 3- to 10-mm-diameter lesions that did not extend into the flesh of the fruit were collected from a field in Frio County, TX, in October 1996. Lesions were either water-soaked or had a scabby center with a water-soaked edge. Non-fluorescent, gram-negative bacteria were consistently isolated from lesions. Eleven representative bacterial strains had 0.835 to 0.950 similarity to Acidovorax avenae subsp. citrulli, using Biolog GN Microplates and the MicroLog data base release 3.50 (Biolog, Hayward, CA). Strains were tested for pathogenicity on watermelon (C. lanatus (Thunb.) Matsum. & Nakai ‘Royal Sweet’) and honeydew seedlings (cv. Morning Ice) by daubing suspensions (approximately 108 CFU/ml) of bacteria onto cotyledons of 1-week-old transplants. Water soaking, followed by necrosis, occurred after 3 to 5 days. A. avenae subsp. citrulli, the causal agent of bacterial fruit blotch of watermelon, was reisolated from lesions. Detached fruit of honeydew (various cultivars) and watermelon (cv. Emperor) were surface disinfested with 70% ethanol, injected sub-epidermally with bacterial suspensions in blemish-free areas, and incubated at 24°C. Water-soaked lesions developed on fruit 10 to 14 days later and the bacteria were reisolated from inoculated fruit. Fruit inoculated with sterile water did not develop symptoms. Female flowers of honeydew plants (cv. Honeybrew) were inoculated at the time of pollination with a suspension of bacteria daubed on the fruit surface. Pollinated fruits were sealed in a moist chamber for 48 h. Circular, water-soaked spots developed 10 to 14 days later. A. avenae subsp. citrulli was reisolated from these lesions. The affected 44-ha field utilized center pivot irrigation and the incidence of diseased fruit exceeded 50%. The source of the pathogen is unknown. Bacterial fruit blotch was identified in watermelon grown under overhead irrigation 0.8 km from this honeydew field 9 weeks earlier. In past years, bacterial fruit blotch has occurred in watermelon fields in Frio County (1). Honeydew from other fields in the vicinity, including one that was also sprinkler irrigated, did not show these symptoms. This is the first report of A. avenae subsp. citrulli causing a disease of honeydew fruit. Reference: M. C. Black et al. Plant Dis. 78:831, 1994.

The bacterium causing fruit blotch of watermelon was transmitted in seeds from fruit that had the typical water-soaking symptom; from fruit that had small, restricted, necrotic lesions; and from symptomless fruit adjacent to typically symptomatic fruit. Storage of seed at 12°C for 12 months did not reduce the level of seed transmission. Fermentation of seeds in watermelon juice and debris prior to washing and drying reduced the level of seed transmission from symptomatic fruit from 61% to less than 1%. Treatment of washed seeds from symptomatic fruit with 1% CaOCl 2 for 15 min was relatively ineffective in reducing seed transmission, but 1% HCI for 15 min was as effective as fermentation. Fermentation of seeds for 24 to 48 h followed by 1% HCI or 1% CaOCl 2 treatment for 15 min prior to washing and drying were the most effective treatments for eliminating bacterial contamination of watermelon seeds. These treatments did not adversely affect seed germination.

Bacterial fruit blotch, a recently described disease of watermelon, resulted in severe losses to Indiana watermelon growers in 1989. Strains of the pathogen recovered from symptomatic fruit and seedlings exhibited similar phenotypic characteristics. Seedling symptoms (water-soaked lesions becoming necrotic on cotyledons and true leaves) and fruit symptoms (large, watersoaked blotches on the exposed rind surface) were induced by strains from both sources. Seed transmission was demonstrated in both seed from symptomatic fruit and seed soaked in a suspension of the bacterium(.)

Mature watermelon fruit with large, firm, dark green, water-soaked lesions were observed in spring 1989. Losses of up to 50% in marketable fruit occurred in some fields in Florida. A gram-negative, aerobic, rod-shaped, nonfluorescent, oxidase-positive, arginine dihydrolase-negative bacterium was isolated from diseased tissues. In pathogenicity tests, the bacterium produced symptoms in foliage and fruit. Morphological, physiological, and biochemical properties of the bacterium related it to a previously described pathogen of watermelon, Pseudomonas pseudoalcaligenes subsp. citrulli

Ten nonfluorescent Pseudomonas strains isolated from water-soaked lesions on cotyledons of plants of five Citrullus lanatus (watermelon) plant introductions were characterized and compared phenotypically with 22 other pseudomonads. The strains were distinguished phenotypically from other known plant pathogenic pseudomonads. The watermelon bacterium was aerobic. Cells were rod-shaped, gram negative, and motile by means of a single polar flagellum. They were nonfluorescent and grew at 41°C but not at 4°C. Oxidase production and the 2-ketogluconate reaction were positive. The 10 strains utilized β-alanine, L-leucine, D-serine, n-propanal, ethanol, ethanolamine, citrate, and fructose for growth. No growth occurred with sucrose or glucose. Their deoxyribonucleic acid base composition was 66 ± 1 mol% guanine plus cytosine. The bacterium is phenotypically similar to P. pseudoalcaligenes but differs from it in being pathogenic to watermelon, Cucumis melo (cantaloupe), Cucumis satiwus (cucumber), and Cucurbita pepo (squash). The name P. pseudoalcaligenes subsp. citrulli is proposed for the new subspecies, of which strain C-42 (= ATCC 29625) is the type strain.