Genetic resistance to bacterial blight disease in Persian walnut

Bacterial blight disease of Persian walnut (Juglans regia, L.), caused by Xanthomonas arboricola pv. juglandis (Xaj), leads to significant nut losses in northern, central and western areas of Iran. To identify the natural sources of resistance to disease in the endemic walnut genotypes of Iran, sixteen walnut genotypes, collected from different areas of Hamedan province, were inoculated with Xaj in a randomized complete block design with five replicates for each genotype. Two-year old genotypes were gently sprayed with a suspension of bacteria adjusted to approximately 2 × 10⁹ cfu ml⁻¹ of distilled water in May. Infected leaves were rated for disease 28 and 42 days after inoculation, using a 0 to 5 severity scale, based on the number, size and distribution of lesions on the leaves. Data analyses showed that there were variations among genotypes in response to pathogen. Upon inoculation by bacterial suspension genotype 94 showed the highest resistance to both disease incidence and its progress after 4–6 weeks of infection. Genotype 65 showed high susceptibility to disease and genotype 69 showed high susceptibilities both to disease incidence and its progress after 4–6 weeks of infection.     

The genetic characterization of Xanthomonas arboricola pv. juglandis,the causal agent of walnut blight in Poland

Leaves and fruits of walnut trees exhibiting symptoms of bacterial blight were collected from six locations in Poland. Isolations on agar media resulted in 18 bacterial isolates with colony morphology resembling that of the Xanthomonas genus. PCR using X1 and X2 primers specific for Xanthomonas confirmed that all isolates belonged to this genus. In pathogenicity tests on unripe walnut fruits, all isolates caused typical black necrotic lesions covering almost the entire pericarp. Results of selected phenotypic tests indicated that characteristics of all isolates were the same as described for the type strain of Xanthomonas arboricola pv. juglandis. Genetic analyses (PCR MP, ERIC‐, BOX‐PCR and MLSA) showed similarities between the studied isolates and the reference strain of X. arboricola pv. juglandis CFBP 7179 originating from France. However, reference strains I‐391 from Portugal and LMG 746 from the UK were different. MLSA analysis of partial sequences of the fyuA, gyrB and rpoD genes of studied isolates and respective sequences from GenBank of pathotype strains of other pathovars of X. arboricola showed that the X. arboricola pv. juglandis isolates consisted of different phylogenetic lineages. An incongruence among MLSA gene phylogenies and traces of intergenic recombination events were proved. These data suggest that the sequence analysis of several housekeeping genes is necessary for proper identification of X. arboricola pathovars.     

Regulation of defense-related enzymes associated with bacterial spot resistance in Tomato

Twenty tomato (Solanum lycopersicon) cultivars were screened for resistance against bacterial spot disease incited byXanthomonas axonopodis pv.vesicatoria under field conditions with and without pathogen infection. Screening was done by artificially inoculating aX. axonopodis pv.vesicatoria suspension to 4-week-old tomato seedlings and observing them for typical symptoms of the disease. Seedlings were categorized into highly resistant, resistant, susceptible and highly susceptible cultivars on the basis of disease incidence. Tomato cultivars were screened for defense-related enzymes, total phenols and lignin contents. The temporal patterns of all these enzymes were estimated with a moderately susceptible tomato cultivar. Native PAGE analysis of both peroxidase (POX) and polyphenol oxidase (PPO) was carried out for the time course of enzyme activities and also by selecting three different tomato cultivars, following infection with the pathogen. Based on the inducible amounts of these enzymes upon pathogen infection, the tomato cultivars were correlated with the disease incidence under field conditions. A significant (P≤0.05) correlation was observed between the degree of host resistance and the enzyme levels. In highly resistant tomato cultivars the enzyme levels, total phenols and lignin contents were increased in comparison with highly susceptible tomato cultivars. Isoform analysis of POX and PPO enzymes indicated a clear difference between resistant and susceptible tomato cultivars in the number of isoforms and also in the intensity of each isoform in the presence of pathogen infection. The possible regulation of defense-related enzymes in imparting host resistance is discussed. http://www.phytoparasitica.org posting March 11, 2008.     

Identification of the anthracnose fungus of walnut (Juglans spp.) and resistance evaluation through physiological responses of resistant vs. susceptible hosts

Walnut anthracnose is one of the most serious diseases of walnut globally. In this study, the pathogen was identified as Colletotrichum gloeosporioides by morphological and molecular approaches. Then, the resistance of 18 walnut genotypes was evaluated by field investigation as well as field and detached leaflet inoculation. A field inoculation assay was used as a benchmark for comparing field evaluation and the detached leaflet inoculation assay. Seven genotypes, Panhe1 and clones 71 and 200 of Juglans sigillata, Yanyuanzao and Qingxiang of J. regia, and Shujiang1 and Chuanzao1 of J. regia × J. sigillata, displayed high resistance in all assays, suggesting that they may be good candidates for further evaluation. Subsequently, two genotypes (Panhe1 with high resistance, and clone 199 with high susceptibility) were selected to analyse the different physiological responses between highly resistant and susceptible genotypes after inoculation. C. gloeosporioides infection induced the activities of superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO), and chitinase; they were all significantly increased in both Panhe1 and clone 199. However, the POD activity and its change rate in Panhe1 were significantly higher than those in clone 199. The change rates of PPO activity, and total phenol and proline content in Panhe1 were significantly higher than those in clone 199. Moreover, the change rates of SOD activity, malondialdehyde, and soluble sugar content in clone 199 were significantly higher than those in Panhe1. These findings not only enhance our understanding of the resistance of walnut to anthracnose, but also lay the foundation for breeding anthracnose-resistant walnuts in the future.     

Identification of a genetic lineage within Xanthomonas arboricola pv. juglandis as the causal agent of vertical oozing canker of Persian (English) walnut in France

A new bacterial disease of Persian (English) walnut (Juglans regia) has been observed in France. This disease, called vertical oozing canker (VOC), is characterized by vertical cankers on trunks and branches of affected walnut trees with oozing exudates. To determine the aetiology of the disease, a study was carried out in 79 walnut orchards and nurseries located in southeastern and southwestern France. Bacterial analysis from diseased samples yielded 36 strains identified as Xanthomonas arboricola and 32 strains identified as Brenneria nigrifluens on the basis of biochemical tests. The causal agent of VOC was identified as X. arboricola by pathogenicity tests on walnut. Fluorescent amplified fragment length polymorphism (F‐AFLP) was carried out on 36 strains of X. arboricola collected in this study, 24 strains of X. arboricola pv. juglandis isolated from walnut blight symptoms and one strain of X. arboricola pv. corylina included as an outgroup. Based on cluster analysis of F‐AFLP data, most X. arboricola strains responsible for main VOC outbreaks showed a high degree of similarity, forming a cluster clearly separate from strains of X. arboricola pv. juglandis isolated from walnut blight symptoms. It is suggested that VOC is caused by a distinct genetic lineage within the pathovar juglandis of X. arboricola that is also able to cause classical bacterial blight symptoms on walnut leaves and fruits.     

Identification of Resistance to Common Bacterial Blight Disease on Bean Genotypes Grown in Turkey

Common bacterial blight (CBB) in edible beans (Phaseolus vulgaris), incited Xanthomonas campestris pv. phaseoli, reduces bean yields and seed quality. The main objective of this study was to determine resistance to common bacterial blight in bean genotypes. Twenty-two bean genotypes grown in Turkey including common and snap bean cultivars/lines were collected from different parts of Turkey and tested for resistance against to Xanthomonas campestris pv. phaseoli strain MFD-11. All the common and snap bean lines/cultivars tested were moderately susceptible, susceptible or highly susceptible, except AG-7117 which was found resistant to Xanthomonas campestris pv. phaseoli. This is the first report of a resistance source in a common bean line (AG-7117) against Xanthomonas campestris pv. phaseoli.     

Polyphasic phenotypic and genetic analysis reveals clonal nature of Xanthomonas axonopodis pv. punicae causing pomegranate bacterial blight

Yellow-pigmented bacteria isolated from blight-affected pomegranate leaves and fruit across seven Indian states in epidemics during the years 2008–2016 were characterized and identified using phenotypic and genotypic tools. All bacterial isolates shared phenotypic traits such as colony morphology, NaCl and pH sensitivity and fuscan production, and caused typical lesions on pomegranate plants upon artificial inoculation. Analysis of 16S ribosomal DNA and 16S–23S rDNA intergenic spacer sequences confirmed their identity as Xanthomonas axonopodis pv. punicae. The new isolates collected after 2000 were compared with an old isolate from the 1950s using polyphasic taxonomic approaches including multilocus sequence analysis (MLSA). Nucleotide polymorphism in 24 isolates for nine genomic loci (dnaK, fyuA, gyrB (Young), gyrB (Almeida), rpoD, fusA, gapA, gltA and lepA) showed minor variations in loci fyuA and gyrB. Isolates were grouped into four nearly identical sequence types, ST1, ST2, ST3 and ST4, based on their allelic profiles, ST3 being widespread in Indian states. Molecular phylogenetic analysis of concatenated 5690 bp with other Xanthomonas pathovars revealed its close genetic similarity with the X. citri group. The blight outbreak in diverse geographical locations is attributed to a re-emerged clonal population of X. axonopodis pv. punicae on a genetically homogenous pomegranate cultivar. The latently infected vegetative planting material of elite pomegranate cultivars contributed to the dissemination of the bacterial inoculum. This study highlights and forewarns of the role played by the clonally propagated elite pomegranate cultivars in disseminating and sustaining clonal populations of this bacterial plant pathogen in many Indian states.     

Notes on the Occurrence of Pathogenic Races of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>Found in Hiroshima Prefecture in 1999

The pathogenic race of 59 cultures of Xanthomonas oryzae pv. oryzae, a pathogen of bacterial leaf blight of rice, isolated from six locations in the inland mountainous area of Hiroshima Prefecture in 1999, were determined by a set of traditional differentials. Four races—I, II, V and VII—were found across the area; however, we noticed the composition of the races as well as the dominant race in each location different. All races were avirulent on differential cultivar Te-tep. Races V and VII were new to Hiroshima. The rice cultivars infected with bacterial leaf blight in Hiroshima are thought to be grouped into the Kinmaze group, which does not have any resistance genes. Apparently, a variety of races occurred unexpectedly on the cultivars contrary to stabilizing selection theory. Received 25 February 2000/ Accepted in revised form 13 July 2000     

Evaluation of glyphosate-tolerant soybean cultivars for resistance to bacterial pustule

Xanthomonas axonopodis pv. glycines causes bacterial pustule of soybean, which is a common disease in many soybean-growing areas of the world and is controlled by a single recessive gene (rxp gene) commonly found in many conventional glyphosate-sensitive soybean cultivars. Since glyphosate-tolerant cultivars are commonly planted today, there has been no information about whether these new cultivars have bacterial pustule resistance. The goal of this study was to screen glyphosate-tolerant soybean cultivars for resistance to X. axonopodis pv. glycines. Three experiments were completed to evaluate resistance. In experiment 1, 525 commercial glyphosate-tolerant cultivars from 2001 were inoculated with X. axonopodis pv. glycines strain UIUC-1. Following inoculation, many of the cultivars were resistant (developed no detectable pustule symptoms) although 152 (~29%) developed bacterial pustule. In experiment 2, the aggressiveness of three strains (UIUC-1, UIUC-2, and ATCC 17915) of X. axonopodis pv. glycines were compared on three bacterial pustule-susceptible, glyphosate-tolerant cultivars. One strain (UIUC-1) was less aggressive than the other two (UIUC-2 and ATCC 17915) on all three cultivars examined. In experiment 3, 45 cultivars from 2005 (all different from 2001) were inoculated with X. axonopodis pv. glycines ATCC 17915. A range of disease severities developed with five cultivars (11%) having disease severity ratings as high as or higher than those on a susceptible check cultivar. Overall, these results suggested that resistance to bacterial pustule occurs in glyphosate-tolerant soybean cultivars, but not at 100% frequency, which means bacterial pustule outbreaks could occur when a susceptible cultivar is planted and conditions are conducive for bacterial pustule development.     

Pathogenicity and virulence gene content of Xanthomonas strains infecting Araceae,formerly known as Xanthomonas axonopodis pv. dieffenbachiae

Bacterial leaf blight of aroids is caused by a heterogeneous group of xanthomonads listed as Xanthomonas axonopodis pv. dieffenbachiae (Xad) on the EPPO A2 quarantine list. Recently, Xad strains were shown not to belong to X. axonopodis but to the species X. citri, X. phaseoli and X. euvesicatoria. Here, to verify the pathovar designation, 11 representative strains were tested for pathogenicity on six aroid genera. They had overlapping host ranges and only the strain isolated from Syngonium showed host specificity. The X. citri strains, isolated from various hosts, showed dissimilarity in virulence to the tested aroid genera. The X. phaseoli strains, isolated from Anthurium and Syngonium, were generally more virulent and, additionally, induced systemic infections. The X. euvesicatoria strains, isolated from Philodendron, were scored as not pathogenic on the tested aroids. Four representative strains were genome sequenced and showed a variable virulence‐associated gene content. Pathogenicity to aroids was correlated with the presence of three specific T3 effector genes and with a T6SS gene sequence. Together, the phylogenetic and pathogenic differentiation among Xad strains justifies the installation of three pathovar epithets for the pathogens on aroids: X. phaseoli pv. dieffenbachiae comb. nov. for the strains isolated from Anthurium; X. phaseoli pv. syngonii comb. nov. for the strain isolated from Syngonium; and X. citri pv. aracearum comb. nov. for the strains isolated from Aglaonema, Xanthosoma and Dieffenbachia. It is proposed that phytosanitary regulations for xanthomonads on aroids are restricted to these three pathovars.     

Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

The present study developed a pathovar‐specific PCR for the detection of Xanthomonas campestris pv. musacearum (Xcm), the cause of banana xanthomonas wilt, by amplification of a 265‐bp region of the gene encoding the general secretion pathway protein D (GspD). A distinct DNA fragment of the expected size was amplified from genomic DNA from all of 12 Xcm isolates tested and no amplification of DNA was observed from other xanthomonads or plant‐associated bacteria, including the two closely related species Xanthomonas vasicola pv. holcicola and Xanthomonas axonopodis pv. vasculorum. The Xcm‐specific PCR was successfully multiplexed with internal control primers targeting 16S rDNA for application on DNA from bacterial cultures and with primers targeting plant mitochondrial 26S rDNA for application on DNA extracted from plant material. Diagnostic discrimination of healthy and infected plants was subsequently demonstrated in tests on artificially inoculated screenhouse cultivars of banana and field bananas with and without symptoms sampled from different parts of Uganda. This study therefore demonstrated a robust and specific Xcm diagnostic tool with the added advantage of applying internal PCR controls for direct quality assessment of results.     

Notes on the occurrence of pathogenic races of Xanthomonas oryzae pv. oryzae found in Hiroshima Prefecture during 2000–2003

The pathogenic races of 450 cultures of Xanthomonas oryzae pv. oryzae, isolated from eight locations in the inland mountainous area of Hiroshima Prefecture during 2000 to 2003, were determined with a set of Japanese differentials. The rice cultivars infected with the bacterium are thought to be in the Kinmaze group, which does not have any resistance genes. Five Japanese races IA, IB, II, V, and VII occurred across the area, although the composition of the races in each location altered during the surveyed 4 years.     

PCR-mediated Detection of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>by Amplification of the 16S–23S rDNA Spacer Region Sequence

A detection method specific for Xanthomonas oryzae pv. oryzae, the pathogen responsible for bacterial blight of rice, was based on the polymerase chain reaction (PCR) and designed by amplifying the 16S–23S rDNA spacer region from this bacterium. The nucleotide sequence of the spacer region between the 16S and 23S rDNA, consisting of approximately 580-bp, from X. oryzae pv. oryzae, X. campestris pv. alfalfae, X. campestris pv. campestris, X. campestris pv. cannabis, X. campestris pv. citri, X. campestris pv. cucurbitae, X. campestris pv. pisi, X. campestris pv. pruni and X. campestris pv. vitians, was determined. The determined sequences had more than 95% identity. Therefore, a pair of primers, XOR-F (5′-GCATGACGTCATCGTCCTGT-3′) and XOR-R2 (5′-CTCGGAGCTATATGCCGTGC-3′) was designed and found to specifically amplify a 470-bp fragment from all strains of X. oryzae pv. oryzae isolated from diverse regions in Japan. No PCR product was amplified from X. campestris pathovars alfalfae, campestris, cannabis, carotae, cucurbitae, dieffenbachiae, glycines, pisi, pruni, vitians or zantedeschiae, except for pathovars citri, incanae and zinniae. The method could also detect the pathogen in infected rice leaves within 3 hr, at a detection limit of 4×10¹ cfu/ml. Received 17 December 1999/ Accepted in revised form 10 April 2000     

Characterization of ISPsy2 and ISPsy3, Newly Identified Insertion Sequences in Pseudomonas syringae pv. eriobotryae

Two new active insertion sequences, ISPsy2 and ISPsy3, were isolated from Pseudomonas syringae pv. eriobotryae, the causal agent of stem cankers of loquat trees. ISPsy2 is 1194-bp long, has 16-bp imperfect terminal inverted repeats, and generates a 4-bp target site duplication upon insertion into the selective cartridge of the entrap vector pSHI1063. The nucleotide sequence of ISPsy2 is completely identical with that of the previously identified IS-like element located adjacent to the virulence gene psvA of Pseudomonas syringae pv. eriobotryae NAE6. The single open reading frame of ISPsy2 encodes a 323-amino-acid protein that has similarity to the transposase of the IS5 subgroup of the IS5 family. The ISPsy3 belonging to the IS91 family is 1507 bp in length, does not duplicate its target sequence, GAAC, and presents an 81% sequence homology with IS801 in P. s. pv. phaseolicola. The transposase of ISPsy3 possesses the conserved amino acid motifs found in the rolling-circle replication protein. Southern blot analysis indicated that multiple copies of ISPsy2 and ISPsy3 are present in the genomes of P. s. pv. eriobotryae and some of the other P. s. pathovars tested. Received 16 August 2001/ Accepted in revised form 19 October 2001     

Growth Complementation of hrpXo Mutants of Xanthomonas oryzae pv. oryzae by Virulent Strains in Rice Cultivars Resistant and Susceptible to the Parental Strain

Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae) T7174 is virulent on rice cultivar IR24 and avirulent on IR-BB2. From recent reports, some virulence and avirulence factors of plant pathogenic bacteria are transferred to plant cells through the hrp-dependent type III secretion system. In this study, we investigated the involvement of hrp genes in the compatible and the incompatible interactions between rice and X. o. pv. oryzae after co-inoculation with hrpXo mutants derived from T7174 and virulent strains. Growth of the mutants, named 74ΔHrpXo and 76ΔHrpXo, was repressed in IR24 when the mutants were applied alone. However, growth of the mutants was complemented by co-inoculation with virulent strains. Growth of bioluminescent hrpXo mutant 76ΔHrpXo in IR24 and its growth in IR-BB2 after co-inoculation with T7133, which is virulent on both cultivars, was equally complemented, as detected by bioluminescence from the mutant. On the other hand, only partial complementation of growth of T7174L76, which is a bioluminescent and pathogenic derivative of T7174, by T7133 was observed in IR-BB2. Thus, growth of the hrpXo mutant of X. o. pv. oryzae was complemented by virulent strains in both susceptible and resistant rice leaves with the parental strain. Received 21 July 2000/ Accepted in revised form 26 October 2000     

<Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">cerasicola</Emphasis>, pv. nov., the Causal Agent of Bacterial Gall of Cherry Tree

Bacterial gall on trunks and twigs of cherry trees (Prunus × yedoens, Someiyoshino) was found in Miyazaki and Saga prefectures, Japan. The surface of young galls are relatively smooth and light brown, but they become rough and dark brown. Characteristics of the bacterium isolated from galls on trunks or twigs are similar to those of Pseudomonas syringae pathovars, i.e., pv. actinidiae, pv. daphniphylli, pv. dendropanacis, pv. Morsprunorum, pv. myricae, pv. rhaphiolepidis, pv. syringae and pv. tremae. This bacterium produced galls on cherry and apricot, but not on 66 other species of plants belonging to 39 families. From these results, this bacterium was classified as a new pathovar of Pseudomonas syringae, and the name Pseudomonas syringae pv. cerasicola, pv. nov., is proposed. Strain M9501(ICMP 13926) was designated as the pathotype strain. Received 10 September 1999/ Accepted in revised form 24 December 1999     

Genomics‐informed design of loop‐mediated isothermal amplification for detection of phytopathogenic Xanthomonas arboricola pv. pruni at the intraspecific level

The objective of this study was to develop a rapid, sensitive detection assay for the quarantine pathogen Xanthomonas arboricola pv. pruni, causal agent of stone fruit bacterial spot, an economically important disease of Prunus spp. Unique targets were identified from X. arboricola pv. pruni genomes using a comparative genomics pipeline of other Xanthomonas species, subspecies and pathovars, and used to identify specific diagnostic markers. Loop‐mediated isothermal amplification (LAMP) was then applied to these markers to provide rapid, sensitive and specific detection. The method developed showed unrivalled specificity with the 79 tested strains and, in contrast to previously established techniques, distinguished between phylogenetically close subspecies such as X. arboricola pv. corylina. The sensitivity of this test is comparable to that of a previously reported TaqMan^? assay at 10³ CFU mL^?1, while the unrivalled speed of LAMP technology enables a positive result to be obtained in <15 min. The developed assay can be used with real‐time fluorescent detectors for quantitative results as well as with DNA‐staining dyes to function as a simplified strategy for on‐site pathogen detection.     

Bacterial Blight of Welsh Onion : A New Disease Caused by Xanthomonas campestris pv. allii pv. nov.

In June of 1998, a new bacterial disease was observed on Welsh onion in Okinawa Prefecture, Japan. Infected plants in nursery boxes were stunted with tip dieback, and heavily infected plants died. In fields, the disease appeared on leaves as irregular gray spots or elliptical spots with creases in the center. These spots enlarged and spread rapidly continued cloudy or rainy weather, and formed blight lesions on outer leaves. Yellow mucoid bacterial colonies were consistently isolated from these lesions. The causal bacterium was identified as a pathovar of Xanthomonas campestris on the basis of bacteriological properties. The bacterium was pathogenic to Welsh onion, onion, but nonpathogenic to chive, Chinese chive and hyacinth. Of Liliaceae plants, which contain Welsh onion and onion, only hyacinth has been reported as a host for the genus Xanthomonas, namely X. campestris pv. hyacinthi. However, strains of X. campestris pv. hyacinthi were not pathogenic against either Welsh onion or onion. From these results, the bacterium isolated from Welsh onion is considered to be a new pathovar of X. campestris, and the name of X. campestris pv. allii pv. nov. is proposed. A strain MAFF 311173 is designated as the pathotype strain. Received 29 March 2000/ Accepted in revised form 4 July 2000     

The tomato Pto gene confers resistance to Pseudomonas floridensis,an emergent plant pathogen with just nine type III effectors

A newly discovered bacterial species, Pseudomonas floridensis, has emerged as a pathogen of tomato in Florida. This study compares the virulence and other attributes of P. floridensis to Pseudomonas syringae pv. tomato, which causes bacterial speck disease of tomato. Pseudomonas floridensis reached lower population levels in leaves of tomato as compared to the P. syringae pv. tomato strains DC3000 and NYT1. Analysis of the genome sequence of the P. floridensis type strain GEV388 revealed that it has just nine type III effectors including AvrPtoB_GEV388, which is 66% identical to AvrPtoB in DC3000. Five of these effectors have been previously reported to be members of a ‘minimal effector repertoire’ required for full DC3000 virulence on Nicotiana benthamiana; however, GEV388 grew poorly on leaves of this plant species compared to the DC3000 minimal effector strain. The tomato Pto gene recognizes AvrPtoB in race 0 P. syringae pv. tomato strains, thereby conferring resistance to bacterial speck disease. Pto was also found to confer resistance to P. floridensis, indicating this gene will be useful in the protection of tomato against this newly emerged pathogen.