Biochemical and molecular detection of Xanthomonas citri subsp. malvacearum,the causal organism of bacterial blight of cotton in the northern cotton zone of Nigeria

Xanthomonas citri subsp. malvacearum causes severe qualitative and quantitative losses to farmers in cotton growing areas of the world. Timely detection of the bacterial pathogen causing blight in cotton is extremely important in developing management strategies against the disease. Bacterial isolates were extracted from cotton seeds obtained from five ginneries located in northern Nigeria. Bacterial colonies were isolated and tested for nitrate reductase activity, oxidase reaction, catalase reaction, aesculin hydrolysis, utilisation of carbon from different sources and Tween 80 hydrolysis was assessed, PCR was also carried out on the isolates. The studied bacteria from the four locations reacted positively to Tween 80 hydrolysis and catalase test. Maltose digestion was negative in all samples, hydrolysis and digestion of arabinose, arabitol, cellobiose, lactate and acetate were positive. They were thus identified as members of the species X. citri based on the biochemical tests run. Molecular detection using PCR positively identified the pathogen.     

Distribution and Races of Xanthomonas axonopodis pv. malvacearum on Cotton (Gossypium hirsutum) in Uganda

Surveys in 1995 and 1996 showed that bacterial blight caused by Xanthomonas axonopodis pv. malvacearum occurs throughout the main cotton growing areas of Uganda, causing seedling blight, angular leaf spot and bacterial boll rot. During the vegetative and early fruiting stages of crop growth, severe symptoms of `blackarm' spread from leaves to the stem, causing loss of fruiting branches. A set of Upland cotton cultivars ( Gossypium hirsutum ) were then used to determine the races of the blight bacterium present in Uganda. Many of the isolates induced moderate to severe symptoms on all the test hosts except 101–102B, indicating infection with race 10 or 18. The next most common isolate was race 7. Races 16 and 6 were also identified and 23% of isolates caused symptoms on all the differential cultivars including 101–102B, results indicating the presence of a race of the pathogen which may be the same as that identified in countries neighbouring Uganda and designated as race 20.     

Assessment of resurgence of bacterial blight and its effect on cotton yield in northern Nigeria

Abstract

Cotton production in Nigeria causes fluctuating socio-economic and biotic factors. Bacterial blight induced by Xanthomonas campestris pv malvacearum causes the greatest yield loss annually. A study was therefore carried out to investigate the resurgence of the disease on 10 different cotton genotypes in Dowaya, Kem and Ngurore which are the major cotton areas of Adamawa state of Nigeria under field conditions. Other objectives were to determine the relationship between the different manifestations of bacterial blight and the yield of seed cotton and to identify resistance in the selected genotypes to the disease manifestations. Results revealed the presence of angular leaf spot, vein and boll rot manifestations of the disease in the study areas. The absence of vein blight symptoms in the Kem location did not result in higher yield because the severity of the leaf spot for this location was relatively higher than for the other two locations. Results also revealed that despite the high severity of angular leaf spot, high boll rot and vein blight incidences observed particularly on SAMCOT-11, SAMCOT-13 and ex-Benin in these locations with a high yield of seed cotton was recorded. The three multi-adversity resistant genotypes (MAR), TX-CDP37HH-1-83, TAMCOT SP-21S and TAMCOT CAMD-E recorded relatively lower severity and incidences in both cases. There was, however, a significantly negative correlation between angular leaf spot severity, boll rot incidence and yield at Kem location, significant negative correlation between angular leaf spot severity, boll rot incidence, vein blight and yield at Dowaya location, as well as significant negative correlation between angular leaf spot severity, vein blight and yield at Ngurore location. The tolerant and high yielding SAMCOT-11, SAMCOT-13 and ex-Benin should through breeding work be improved to be used for production in this area and its surroundings.     

Cocculus hirsutus extract inhibits the Xanthomonas oryzae pv. oryzae,the bacterial leaf blight pathogen in rice

Plant-derived natural bactericides and their possible applications in agriculture to control plant bacterial diseases has intensified as this approach has enormous potential to inspire and influence modern agro-chemical research. Naturally occurring and biologically active plant products such as essential oils and organic extracts could be a source of alternative classes of natural biopesticides to serve as templates for new and more effective compounds in controlling plant pathogenic micro-organisms. In the present study, the efficacy of six plants extracts from different solvent system were tested for their antibacterial activity aganist Xanthomonas oryzae pv. oryzae both in vitro and in vivo. Among these extracts, Cocculus hirsutus leaf chloroform extract exhibits significant antibacterial activity against X. oryzae pv. oryzae. Data obtained from the experiments such as minimum inhibitory concentration, effect of C. hirsutus leaf chloroform extract on the incidence of X. oryzae pv. oryzae, phytotoxicity test and effect of C. hirsutus leaf chloroform extract on seed germination and seedling vigour, along with the in vivo experiments under greenhouse conditions showed significant improvement over controls. Thus, the present study demonstrated that the C. hirsutus leaf chloroform extract posses antibacterial activity against bacterial leaf blight pathogen of rice.     

Biocontrol of citrus bacterial canker caused by Xanthomonas citri subsp. citri by Bacillus velezensis

Microorganisms with biocontrol capabilities against plant pathogens are considered as one of the most promising approaches for healthy crop management. In this study, ethyl acetate extracts of 25 Bacillus strains were investigated for their antagonistic effect on Xanthomonas citri subsp. citri (Xcc), which causes the citrus bacterial canker (CBC) disease. Among them, 21 strains exerted antibacterial activity against wild-type Xcc strains. Based on the strength of the antibacterial activity, nine Bacillus strains were selected for 16S rRNA analysis. 16S rRNA sequence homology revealed that several strains were closely related to B. velezensis, where strains with no antibacterial activity grouped as the soil-associated community of B. amyloliquefaciens. B. velezensis Bv-21 exhibited the highest antibacterial activity against wild type and streptomycin resistant Xcc with inhibition zones of 22.91 ± 0.45 and 20.28 ± 0.53, respectively. Furthermore, B. velezensis Bv-21 strain was tested for biocontrol activity against a streptomycin-resistant XccM4 in detached susceptible citrus leaves. The strain reduced the incidence of CBC by 26.30% and pathogen density of XccM4 by 81.68% over control. The results of the study strongly suggest that B. velezensis can be used as an effective and eco-friendly biocontrol agent either by itself or as an active compound, against both, the wild-type and streptomycin-resistant Xcc.     

Development of pre-detection technique for bacterial blight (Xanthomonas axonopodis pv. dieffenbachiae) disease in Anthurium to produce healthy planting materials

Abstract

Anthurium cut-flowers and potted plants have earned a growing marketing demand both in the local and global markets. Bacterial blight, caused by Xanthomonas axonopodis pv. dieffenbachiae has been of major economic concern among the growers worldwide as the disease could cause heavy losses on Anthuriums and other members of the family Araceae. The disease has the potential of spreading latently exhibiting no symptoms through a range of hosts and this fact makes it a prerequisite to detect the presence of pathogen early. No reliable control method so far has been developed and therefore maintenance of strict crop sanitary measures is of great importance. 100 samples representing 30 nurseries were selected from three of the major producing districts, Gampaha, Kurunegala and Puttlam districts. The pathogen was isolated from the samples using 5% KOH test and yeast dextrose calcium carbonate medium. Isolated pathogen was inoculated to healthy Dieffenbachia cane and leaf slices and after 1 – 2 days, symptoms first developed in cane cuttings while leaves took three days for the development of symptoms. Young, tender leaf and cane slices were observed to be highly susceptible to disease. The mature cane slices and leaf cuttings were found to be resistant to the pathogen, most probably due to the non-specific mechanical impermeability of the tissues. Tender leaf and cane tissues, with their ability to get infected and develop visual symptoms relatively fast, worked best with the technique as they proved to shorten the time taken for the detection. The validity and the precision of the pathogen identification test and therefore, the method, were assessed with an indirect-ELISA pathogen-specific detection step, prior to inoculation of healthy tissues. Pathogen was detected to present in 15 samples from Kurunegala and Gampaha districts. The method confirmed its precision, reliability, cost-effectiveness and application under normal laboratory conditions.     

柑橘溃疡病菌II型分泌通道基因xpsD突变体的鉴定及功能分析

【目的】鉴定柑橘溃疡病菌胞外水解酶减弱突变体Mxac56-20的Tn5插入位点,及其在柑橘上的致病力。【方法】采用质粒拯救方法获得Tn5旁侧序列,与基因组信息比对后明确突变体的插入位点;构建功能互补载体对突变体进行功能互补,检测互补菌株胞外蛋白水解酶、纤维素酶和淀粉酶的恢复情况;在寄主植物柑橘上观察致病力变化。【结果】Mxac56-20的Tn5插入位点是II型分泌系统xpsD基因,所构建的互补载体使突变体的胞外水解酶活性和致病力得到恢复。【结论】柑橘溃疡病菌xpsD基因的突变,导致胞外水解酶活性降低,在寄主上的致病力减弱,说明柑橘溃疡病菌的II型分泌系统在与寄主互作过程中起到致病因子的作用。     

Induction of bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in rice by treatment with acibenzolar-S-methyl

The role of the plant defence activator, acibenzolar‐S‐methyl (ASM), in inducing resistance in rice against bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) was studied. Application of ASM induced resistance in rice to infection by Xoo. When the pathogen was clip‐inoculated to the rice plants, it caused bacterial leaf blight symptoms in the untreated control. However, in the rice plants pretreated with ASM, infection was significantly reduced. Induced systemic resistance was found to persist for up to 3 days in the pretreated rice plants. Increased phenolic content and accumulation of pathogenesis‐related (PR) proteins, viz. chitinase, β‐1,3‐glucanase and thaumatin‐like protein (TLP; PR 5) were observed in rice plants pretreated with ASM followed by inoculation with Xoo. Immunoblot analysis using rice TLP and tobacco chitinase antiserum revealed rapid induction and over‐expression of 25 and 35 kDa TLP and chitinase, respectively, in rice in response to pretreatment with ASM followed by Xoo inoculation. Based on these experiments, it is evident that induction of disease resistance in rice was accelerated following treatment with ASM.     

Rapid screenhouse assessment of bacterial blight in cassava genotypes in Nigeria

Bacterial blight disease caused by Xanthomonas axonopodis pv. manihotis (Berthet-Bondar) Dye was assessed in 11 artificially inoculated cassava genotypes in a screenhouse. Disease progress was estimated at intervals of 3 days by measuring the length of necrotic lesions on stems and leaves, as well as estimating the average disease score and area under disease progress curve (AUDPC). Based on the average disease scores, cassava genotypes 30572, TME 1, TME 7 and TME 9 were classified as resistant to bacterial blight, genotypes 4(2)1425, TME 2, TME 4 and TME 12 were tolerant while cassava genotypes 30001, TME 3, and TME 28 were susceptible. Direct correlations, statistically significant at p < 0.05, were obtained between stem necrosis, leaf necrosis, average disease scores and AUDPC in the 11 cassava genotypes. Screenhouse experiments afford rapid assessment of resistance status of cassava genotypes to bacterial blight in Nigeria.     

A 13‐lipoxygenase is Expressed Early in the Hypersensitive Reaction of Cotton Plants to Xanthomonas campestris pv. malvacearum

Lipoxygenases (LOXs) are enzymes responsible for lipid peroxidation processes during plant defence responses to pathogen infection. Jasmonates are lipid‐derived signals that mediate plant stress responses with chloroplastic LOXs implicated in the biosynthesis of oxylipins like jasmonic acid (JA). Hypersensitive reaction (HR) cell death of cotton to the incompatible race 18 of Xanthomonas campestris pathovar malvacearum (Xcm) is associated with 9S‐lipoxygenase activity and expression of a 9‐LOX GhLOX1. Here, we report the cloning of cotton (Gossypium hirsutum L.) LOX gene GhLOX2. Sequence analysis showed that GhLOX2 is a putative 13‐LOX with a chloroplast‐transit peptide in the amino acid terminus. GhLOX2 was found to be significantly expressed in the first hour of Xcm‐induced HR. Investigation into LOX signalization on cotyledons incubated with methyl‐jasmonate (MeJA) or infiltrated with salicylic acid (SA) or ethylene (ET) revealed that the first two treatments induced GhLOX2 gene expression. Our results show that GhLOX2 gene expression occurred at the stage of the HR prior biochemical events previously highlighted. The role that GhLOX2 may have in the defence strategy of cotton to Xcm is discussed regarding the HR.     

Surface layers ofXanthomonas malvacearum,the cause of bacterial blight of cotton

Mureins were isolated from two strains ofXanthomonas malvacearum, a phytopathogenic bacterium causing bacterial blight of cotton. The purity of murein was 70–95 % and the amino acid and amino sugar components (glutamic acid, alanine, meso-diaminopimelic acid, muramio acid and glucosamine) were present at the molar ratio of 1: 1.9: 1: 1.12: 0.85. The bacterium secreted a copious amount of slime which masked its surface structure. The slime was composed of densely interwoven network of filamentous material originating from the cell surface and extended into the medium without any discernable boundary. The slime was secreted through surface layers pores by force, giving the effect of a spray or jet. Slime also played a role in chain formation of bacterial cells.     

RFLP mapping and race specificity of bacterial blight resistance genes (QTLs) in rice

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Detection, Survival and Transmission of Xanthomonas axonopodis pv. manihotis and X. axonopodis pv. vignicola, Causal Agents of Cassava and Cowpea Bacterial Blight, respectively, in/by Insect Vectors

Populations of Xanthomonas axonopodis pv. manihotis and X. axonopodis pv. vignicola, causal agents of cassava and cowpea bacterial blight, respectively, were quantified in insects. The pathogens were found in the faeces, the intestines, and on the legs and mandibles of Zonocerusvariegatus. Additionally, X. axonopodis pv. manihotis was localized in the insect gut by immunofluorescence microscopy. Xanthomonas axonopodis pv. manihotis survived at least 1 week in the insect intestines and at least 5 weeks in faeces kept under controlled conditions, while survival in faeces exposed to sunlight was <2 weeks. Five percentage [e.g. 5.8 × 10⁷ colony‐forming units (CFU)/g faeces] of the fed population of X. axonopodis pv. manihotis in cassava leaves were recovered viable in the faeces after passage through the insect. The transmission of cassava bacterial blight by pathogen‐contaminated insect faeces to intact, healthy cassava leaves was demonstrated for the first time. Xanthomonas axonopodis pv. vignicola was isolated from organs and faeces of the grasshopper Pyrgomorpha cognata, the Senegalese grasshopper (Oedaleus senegalensis), bee (Apis mellifera) and three Coleoptera (Ootheca mutabilis, Mylabris spp., Exochomus troberti) collected in bacterial blight‐infected cowpea fields. Cowpea belonged to the diet of 19 grasshopper species collected in cowpea fields as demonstrated by residues in their faeces. Pathogen‐contaminated Z. variegatus initiated an epiphytic population of 8.9 × 10⁴ CFU/g on healthy cowpea leaves. Spraying cassava and cowpea leaves with 10² and 10⁴ CFU/ml of their respective pathogen was sufficient to evoke symptoms. A possible role of insects in the transmission of X. axonopodis pvs. vignicola and manihotis is discussed.     

Resident population of Xanthomonas campestris pv. malvacearum on cotton leaves: a source of inoculum for bacterial blight

Xanthomonas campestris pv. malvacearum was transmitted from infested seed to the cotyledons of cotton cv. Deltapine 61 seedlings at 28°C and relative humidities (RH) of 90% or 73%. A resident population was present on the first and second true leaves but not on the third true leaf of plants at either RH. There were smaller numbers of resident bacteria on fewer leaves of plants at the lower RH than on plants at the higher RH. Cotton plants grown from infested seed at 25°C and 30°C and incubated at 100% RH at different stages of growth developed bacterial blight on leaves that were in bud or partly expanded when incubated. Resident cells of this pathogen can thus invade susceptible leaves when conditions are favourable for infection. Bacterial blight developed on more plants at 30°C than at 25°C. In a field trial, X. campestris pv. malvacearum transmitted from seed was present as resident bacteria on the third leaf from the growing point during the vegetative development of the plant. Resident bacteria, which infected young leaves during rainy periods, were isolated from the bacterial blight lesions which subsequently developed.     

Effects of the relationship between area under disease progress curve of angular leaf spot and yield of seed cotton in northeastern Nigeria

Bacterial blight induced by Xanthomonas campestris pv. malvacearum causes substantial yield losses of cotton in Nigeria annually. A field study was carried out for two seasons to investigate the reaction of a selection of 10 different cotton genotypes to bacterial blight and to examine the relationship between the area under disease progress curve (AUDPC) and the yield of seed cotton in the northeastern cotton region of Nigeria. All 10 genotypes reacted positively to the disease with SAMCOT-13 having the highest incidence particularly at Dowaya and Kem locations during the two seasons. This investigation also revealed that SAMCOT-13 and Ex-Benin had the highest AUDPC while TX-CDP37HH-1-83, TAMCOT CAMD-E and TAMCOT SP-21S had the lowest AUDPC indicating relative susceptibility and resistance, respectively. However, it was observed that TX-CDP37HH-1-83, SAMCOT-11 and Ex-Benin had the highest mean yields of 1441.5 kg/ha and 1494.7 kg/ha at Dowaya, Kem and Ngurore, respectively. The results also show a highly significant (P = 0.01) negative correlation between AUDPC and yield in the three locations. These findings suggest that TX-CDP37HH-1-83, TAMCOT CAMD-E and TAMCOT SP-21S are moderately resistant to the disease in view of their lower AUDPC and are considered promising.     

Efficacy of different copper compounds in the control of Xanthomonas citri subsp. citri pathotypes A and A*

Citrus canker disease is one of the most devastating diseases that attacks citrus, especially limes in the Southern parts of Iran, and is caused by Xanthomonas citri subsp. citri (Xcc). The efficacy of several formulations of copper compounds including Bordeaux mixture, copper oxychloride and copper sulphate in controlling Xcc in Key lime was estimated in vitro and in planta using artificial inoculation. Specific primers were used to detect copper-resistant genes copA, copB and copL in 30 isolates of Xcc. The copA and copL genes were present in all isolates, and copB was detected only in 6 strains. In this study, we observed a very good in vitro growth inhibition activity of copper compounds against Xcc pathotype A. S14 strain (pathotype A^*) was the sole isolate that grew on media amended with 2/4 mM of Bordeaux mixture, copper oxychloride and copper sulphate. All other strains (pathotype A) failed to grow on media amended with this concentration. Bordeaux mixture exhibited high efficacy in controlling Xcc in both conditions. However, there were no significant differences in the efficacy of copper oxychloride and copper sulphate at 1.2 mM concentration in planta. A significantly minimum canker necrotic spot and highest disease control was achieved with Bordeaux mixture and copper oxychloride. There was a significant difference in disease severity of the type strain LMG9322 (pathotype A) and Xcc strain S14 (pathotype A^*). Our experiments showed that Bordeaux mixture exhibited satisfactory efficacy in controlling the causal agent of citrus canker.     

利用激光共聚焦扫描显微镜对溃疡病菌侵染柑橘叶片的实时观察

柑橘溃疡病对柑橘产业造成了巨大损失,而研究柑橘与溃疡病菌的互作关系以及柑橘的感病和抗病性均需要观察溃疡病菌在柑橘寄主中的侵染和定殖过程。激光共聚焦扫描显微镜不仅可以观察活细胞,活组织的动态代谢过程,而且可以获得三维图像,对于病原菌在柑橘植物组织内的繁殖和致病机制研究具有重要意义。但是,选择适宜的植物材料和制片方法对激光共聚焦扫描显微镜的观察效果影响很大。本文对激光共聚焦扫描显微镜所观察的材料在其处理和观察方法上加以改进,获得了质量更好的图片和实验结果,也使得实验更为方便快捷。激光共聚焦扫描显微观察还在瞬时表达分析中得到应用,提高了柑橘瞬时表达分析的效果。通过将切片和压片相结合观察到溃疡病菌在不同时间点对柑橘叶片的侵染情况,而通过3D建模能观察到柑橘叶片不同组织层面中的病菌数量和病菌位置,为研究溃疡病菌在叶片中的定殖方式和入侵数量提供了前期基础。