Identification and characterization of Xanthomonas albilineans causing sugarcane leaf scald in China using multilocus sequence analysis

Xanthomonas albilineans is the causal agent of leaf scald, a disease that can cause considerable damage to sugarcane industries. This study analysed the phylogenetic relationship of 14 samples of X. albilineans from China and 13 reference strains retrieved from the GenBank database by multilocus sequence analysis (MLSA). To reach this goal, five housekeeping genes of X. albilineans were amplified from diseased leaves and sequenced: gyrB, abc, rpoD, atpD and glnA. Based on the concatenated sequence of these genes (4473 nt), the 14 samples of X. albilineans from China had 99.9–100% sequence identity with one another and with five strains of the pathogen from the French West Indies and the USA (Florida). The 27 samples or strains of X. albilineans were distributed in two distinct clades in the MLSA-based phylogenetic tree. Clade 1 was formed by four strains of the pathogen from Fiji, Papua New Guinea and the USA. All the other strains from worldwide locations, including the 14 samples from China, were grouped in clade 2. This latter clade included all strains of the pathogen that were associated with outbreaks of leaf scald that have occurred over the last two decades, especially in the Caribbean islands and the USA. The very low diversity of X. albilineans in four Chinese provinces suggests recent spread of a single strain (from genetic group PFGE-B) of the leaf scald pathogen within China.     

甘蔗白条病及其致病菌Xanthomonas albilineans研究进展

由白条黄单胞杆菌Xanthomonas albilineans引起的甘蔗白条病是一种寄生在植物维管组织的系统性细菌病害,在全球多数种植甘蔗的国家或地区普遍发生,对甘蔗产业的发展构成潜在威胁。本研究综述了甘蔗白条病的发生和分布、传播与流行规律以及病原菌生物学与基因组特性、鉴定与检测、遗传多样性和致病机理等方面内容;提出加强抗病种质挖掘与新品种选育推广、加快抗病分子育种进程、切断病害传播途径、加强隔离检验检疫等病害防控策略;此外,评估了该病害在我国蔗区流行暴发的风险,展望今后甘蔗抗病分子育种、病原菌致病机制及其与寄主互作机理等方面的分子基础研究重点与应用前景。     

Ultrastructural Changes and Production of a Xanthan-Like Polysaccharide Associated with Scald of Sugarcane Leaves Caused by Xanthomonas albilineans

Leaf scald is a vascular disease of sugarcane caused by Xanthomonas albilineans. Scalded leaves show white-yellowish streaks alternating with green zones in parallel to the main veins. These zones develop large bulliform cells, probably as a consequence of the wilting process. Moreover, a gum exudate occludes phloem and bundle vessels, and partially enters mesophyll cells. Some lysigenic cavities appear near the xylem. However, the white-yellowish streaks show both phloem and xylem completely occluded by the gum and the overall mesophyll appears to be full of this bacterial secretion, as revealed by scanning electron microscopy. The gum in conducting tissues has been purified from juice obtained from scalded stalks by precipitation with isopropyl alcohol and size-exclusion chromatography. It was identified as a xanthan-like polysaccharide and found to be composed of glucose, mannose and glucuronic acid by acidic hydrolysis and capillary electrophoresis.     

Leaf scald (Xanthomonas albilineans) incidence and its effect on yield in seven sugarcane cultivars in Guadeloupe

Seven 5—month-old sugarcane cultivars difTering in resistance to leaf scald disease were inoculated by the decapitation technique with Xanthomonas albilineans. The effects of disease progress and incidence on yields were studied for the plant (first harvest) and two ratoon crops (second and third harvest). The percentage of diseased stalks and disease severity at first harvest 5 months after inoculation were 0·7 and 0·4, respectively, for the most resistant cultivar and 71·0% and 63·3, respectively, for the most susceptible cultivar. They decreased in all cultivars in both ratoon crops, but were still important in one cultivar (B69379). Significant ( P = 0·05) yield reductions of 12% and 21% occurred in two of the seven cultivars (B69566 and B69379, respectively). The number of symptomatic sugarcane stalks in the first ratoon crop (second harvest) was lower than the number of stalks colonized by the pathogen. Symptoms and yield losses of cultivars R570 and B69566 varied with the crop. Yield losses occurred in cultivar R570 only in the plant crop when this cultivar displayed numerous symptoms. Cultivar B69566 appeared to recover from the disease to a certain extent from the plant to the second ratoon crop (third harvest), as did the resistant cultivars in the first ratoon crop. In contrast, severe leaf scald symptoms were observed in the case of cultivar B69379 regardless of the crop, and significant yield losses occurred in the two ratoon crops. These results support the recommendation that cultivar B69379 should not be replanted in Guadeloupe.     

Intraspecific Genomic Variation Within Xanthomonas albilineans, the Sugarcane Leaf Scald Pathogen

ABSTRACT To better understand the nature of recent outbreaks of leaf scald disease of sugarcane in a number of sugarcane production regions of the world including Florida, Guadeloupe, Louisiana, Mauritius, Taiwan, and Texas, a study of the worldwide genetic variation of the pathogen was undertaken. A total of 218 strains from 31 geographic locations were examined. Genomic DNA of each strain was digested with the rare cutting restriction enzyme SpeI, and the fragments were separated by pulsed-field gel electrophoresis (PFGE). A total of 102 bands were identified, and 54 different DNA banding patterns (haplotypes) were observed. Eight groups of banding patterns, designated PFGE groups A through H, were consistently detected by visual, principal component, and cluster analyses. Five groups were comprised of multiple haplotypes representing numerous strains, and three were comprised of single haplotypes representing one strain each. The leaf scald outbreaks in Florida, Louisiana, Texas, and possibly Guadeloupe and Taiwan could be attributed to the introduction of strains belonging to PFGE group B. When infection by two strains each of the newly introduced strains (PFGE group B) and those previously present in Florida (PFGE group A) was analyzed in 22 sugarcane cultivars by reisolation 24 weeks after inoculation, a significantly greater mean frequency was detected for PFGE group B strains and no cultivar by PFGE group interaction was observed. Inadvertent dispersal of the pathogen among plants, possibly by means of aerosols or splashing water, was detected in a subsequent experiment. Strains of PFGE group B were recovered from the internal tissues of some plants inoculated with PFGE group A strains and were also found to be epiphytic colonizers of nonsymptomatic, noninoculated plants adjacent to the inoculated plants; whereas strains of PFGE group A were recovered only from plants that had been inoculated with them. Thus, the possibility became more apparent that strain variation might be associated, at least in part, with factors governing plant-to-plant spread of the pathogen in nature.     

Resistance to Leaf Scald Disease Is Associated with Limited Colonization of Sugarcane and Wild Relatives by Xanthomonas albilineans

ABSTRACT A streptomycin- and rifampicin-resistant mutant of Xanthomonas al-bilineans was used to study symptom expression of leaf scald disease (LSD) and colonization of sugarcane (Saccharum spp.) and its wild relatives by this bacterial pathogen. A total of 40 sugarcane cultivars and 15 clones from the Saccharum complex that differed in resistance to LSD were inoculated by a decapitation technique in both field and greenhouse experiments. In the plant crop, disease severity varied between 0 for the most resistant genotypes and 100 for the most susceptible ones. Resistance to LSD was characterized by limited colonization of the host plant by X. albilineans. Although almost all genotypes were colonized by the pathogen, the greatest bacterial population densities were found in the susceptible cultivars. There was a high correlation between disease severity and pathogen population in the apex. Several genotypes exhibited no or slight symptoms even though they were highly colonized in the upper and/or basal nodes of stalks. Two mechanisms, therefore, may play an important role in resistance to LSD: resistance to colonization of the apex, which is characterized by absence of symptoms, and resistance to colonization of the upper and lower parts of the stalk. In contrast, disease severity and pathogen population densities in the first ratoon crop in the field were nil or very low in the stalks, except for the highly susceptible cv. CP68-1026. Sugarcane ratoons, therefore, may recover from the disease after plant cane infection. Nevertheless, because low levels of the pathogen were still detected in some stalks, it is possible that LSD could develop from latent infections if favorable environmental conditions occur.     

Virulence factors analysis of native isolates of Xanthomonas albilineans and Xanthomonas sacchari from Tucumán,Argentina, reveals differences in pathogenic strategies

Xanthomonas albilineans (Xa) and X. sacchari (Xs) are both sugarcane pathogens. Xa is the causal agent of leaf scald disease, but there is limited information about the pathogenicity of Xs. The aim of this work was to study virulence factors of native strains of Xa (Xa32, Xa33, and XaM6) and Xs (Xs14 and Xs15) previously isolated from sugarcane with leaf scald symptoms, to gain insight into the biology of each microorganism. We analysed epiphytic survival, sensitivity to oxidative stress, extracellular degradative enzymes, cell motilities, exopolysaccharide (EPS) characteristics, cell adhesion, biofilm development, and control of stomatal regulation of the five strains. We observed that each species presented similar phenotypes for every factor analysed. Xa strains appeared to be more sensitive to oxidative stress and presented lower epiphytic survival than Xs. All strains presented endoglucanase activity; however, we could only detect protease and amylase activities in Xs strains. Swimming and sliding were higher in Xs, but twitching was variable among species. We also observed that only Xs strains produced a xanthan-like EPS, presented a strong cell adhesion, and structured biofilm. We detected some intraspecific variations showing that higher amounts of EPS produced by Xs14 correlated with its higher sliding motility and its homogenous and more adhesive biofilm. In addition, EPSs of Xs14 and Xs15 presented differences in strand height and acetyl percentage. Finally, we found that strains of both species were able to interfere with stomatal aperture mechanism. All these differences could influence the colonization strategies and/or disease progression in each species.     

Variation in Albicidin Biosynthesis Genes and in Pathogenicity of Xanthomonas albilineans, the Sugarcane Leaf Scald Pathogen

ABSTRACT Total genomic DNA from 137 strains of Xanthomonas albilineans from worldwide locations was hybridized with two DNA probes that together harbor the entire 49-kb albicidin biosynthesis gene cluster and two additional 3-kb genomic regions required for albicidin production. Fourteen haplotypes and two major genetic groups (albicidin [ALB]-restriction fragment length polymorphism [RFLP] A and ALB-RFLP B) were identified, and strains that were isolated after recent outbreaks of leaf scald disease belonged to group ALB-RFLP B. Albicidin genetic diversity was very similar to the previously described genetic diversity of the pathogen based on the whole genome. No relationship was found between variability of albicidin biosynthesis genes and the amount of albicidin produced in vitro by X. albilineans. Leaf scald-susceptible sugarcane cv. H70-144 was inoculated with 20 strains of the pathogen belonging to different ALB-RFLP haplotypes. Among them, 10 strains from Guadeloupe belonged to the same ALB-RFLP group but differed in the amount of albicidin produced in vitro. Strains were distributed in at least three different pathogenicity groups based on symptom severity and pathogen population density in the stalk. These two pathogenicity factors varied concurrently; however, no relationship between variation in albicidin biosynthesis genes, variation in the amount of albicidin produced in vitro, and variation in pathogenicity of X. albilineans was found. Further investigation is necessary to identify other genes involved in pathogenicity of X. albilineans.     

High Variation in Pathogenicity of Genetically Closely Related Strains of Xanthomonas albilineans, the Sugarcane Leaf Scald Pathogen, in Guadeloupe

ABSTRACT Pathogenicity of 75 strains of Xanthomonas albilineans from Guadeloupe was assessed by inoculation of sugarcane cv. B69566, which is susceptible to leaf scald, and 19 of the strains were selected as representative of the variation in pathogenicity observed based on stalk colonization. In vitro production of albicidin varied among these 19 strains, but the restriction fragment length polymorphism pattern of their albicidin biosynthesis genes was identical. Similarly, no genomic variation was found among strains by pulsed-field gel electrophoresis. Some variation among strains was found by amplified fragment length polymorphism, but no relationship between this genetic variation and variation in pathogenicity was found. Only 3 (pilB, rpfA, and xpsE) of 40 genes involved in pathogenicity of bacterial species closely related to X. albilineans could be amplified by polymerase chain reaction from total genomic DNA of all nine strains tested of X. albilineans differing in pathogenicity in Guadeloupe. Nucleotide sequences of these genes were 100% identical among strains, and a phylogenetic study with these genes and housekeeping genes efp and ihfA suggested that X. albilineans is on an evolutionary road between the X. campestris group and Xylella fastidiosa, another vascular plant pathogen. Sequencing of the complete genome of Xanthomonas albilineans could be the next step in deciphering molecular mechanisms involved in pathogenicity of X. albilineans.     

Impact of Sugarcane yellow leaf virus on Sugarcane Yield and Juice Quality in Réunion Island

Sugarcane yellow leaf virus (SCYLV) was first detected in sugarcane of Réunion Island in 1997. A field experiment was undertaken to assess the potential impact of this virus on sugarcane production. The agronomic characteristics of SCYLV-infected plants were compared to those of virus-free plants of three sugarcane cultivars (R570, R577 and R579) which occupy more than 90% of the cultivated sugarcane area on Réunion Island. In the plant crop, significant losses in stalk weight (28%) and in sugar content (11%) were detected for cultivar R577, but not for either of the two other cultivars. In the first ratoon crop, yield reduction was detected for cultivar R577 (37%), but also for cultivar R579 (19%). Cultivar R577 also showed significant losses in sugar content (12%) due to reduced amount and quality of extracted cane juice. No yield reduction was found for cultivar R570, although stalk height and diameter were reduced in SCYLV-infected canes of this cultivar in the first ratoon crop. Leaf yellowing was observed at harvest of plant and ratoon crops when sugarcane was no longer irrigated, and 10–59% of symptomatic stalks could be attributed to the presence of SCYLV. The most severe yellowing symptoms were related to infection of sugarcane by the virus.     

Differentiation of Xanthomonas species Pathogenic to Sugarcane by PCR-RFLP Analysis

The PCR-RFLP of the 16S-23S rDNA spacer region was used to differentiate Xanthomonas species pathogenic to sugarcane. Strains of X. albilineans, X. campestris pv. vasculorum Types A and B, X. sacchari and Xanthomonas sp. from Trinidad, South Africa and India were examined. The amplification products were digested with Alu I, Hae III, Hpa II and Mbo I and the results showed that the different groups of bacterial strains exhibited distinct RFLP patterns for each tested endonuclease, except X. albilineans and X. sacchari which could only be differentiated from each other by the digestion with Hpa II. The results also allowed the separation of X.c. pv. vasculorum Type A from X.c. pv. vasculorum Type B and strongly suggested that the analyzed Xanthomonas sp. strains belong to X. sacchari. Nine X. campestris (pv. not determined) strains included in this study showed identical profiles to X.c. pv. vasculorum Type A group and DNA–DNA hybridization experiments confirmed these results. PCR-RFLP of the 16S-23S rDNA spacer region could be applied as a reliable method for differentiating the xanthomonads pathogenic to sugarcane.     

广西蔗区甘蔗白条病菌的鉴定与致病力分析

甘蔗白条病是一种检疫性病害,在我国多个蔗区均有报道,严重威胁我国甘蔗生产.本研究对采自广西蔗区不同品种(系)的病原菌进行分离、鉴定、保存,并选取其中40份白条黄单胞菌分离物进行遗传多样性分析与接种试验,评价其致病性.根据三磷酸腺苷结合盒转运蛋白等管家基因和Ⅳ型分泌系统virB基因的序列差异和进化树分析,将40个菌株分为...     

Differentiation of Xanthomonas albilineans strains with monoclonal antibody reaction patterns and DNA fingerprints

Thirty-eight strains of the sugarcane leaf scald pathogen, Xanthomonas albilineans , were compared using a series of seven species-specific monoclonal antibodies as well as genomic DNA fingerprint patterns. The strains, which were obtained from 13 countries and states in the USA, were placed in three major groups with approximately eight subgroups based on similarity of their serological reactions. The serological groupings correlated strongly with groupings based on DNA fingerprint patterns. The groups of strains that were genetically and serologically related did not necessarily come from nearby locations but were usually from widely separated regions. Thus, it appears that disease transmission occurred by means other than natural spread. The methods used in this study are useful for comparing relationships among strains of X. albilineans . A combination of the monoclonal antibodies could be used in a serodiagnostic-based method to test for the presence of leaf scald disease.     

A stable Leifsonia xyli subsp. xyli GFP‐tagged strain reveals a new colonization niche in sugarcane tissues

Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli (Lxx), is one of the most economically important diseases of sugarcane worldwide. Because knowledge on the interaction of Lxx with its host at the microscopic level is limited, the development of tools to monitor Lxx during the colonization process could shed new light on the processes that control disease development. In this investigation, a transformation protocol was optimized and a mutant Lxx strain engineered that stably expressed the gfp gene in sugarcane tissues. In vitro, the growth of the mutant did not differ from that of the wild type. Also, plants inoculated with both strains showed comparable growth and development when analysed 180 days after inoculation (dai). Fluorescence microscopy of roots, stalks, meristems and leaf tissues of Lxx‐GFP‐inoculated plants was performed at 180 dai. In the leaves, Lxx‐tagged cells were observed within the xylem vessels as has been described before but, in addition, they were found in a new niche within the host tissues, in the mesophyll and in the bundle sheath cells surrounding the vascular system. This finding indicates that Lxx is able to move from the xylem to the parenchyma of the leaf cells. This first report of an Lxx mutant expressing a heterologous gene revealed that colonization of sugarcane by this pathogen is not limited to the xylem vessels as commonly reported.     

Ralstonia solanacearum preferential colonization in the shoot apical meristem explains its pathogenicity pattern in tomato seedlings

Ralstonia solanacearum causes a lethal bacterial wilt disease in many plants by colonizing the vascular tissues of the hosts. Upon inoculation of tomato seedlings through either leaf or root, the wilting symptoms occur first at the apical region and then proceed downward along the shoot. The systemic order of the disease initiation and progression in the host, independent of the site of pathogen inoculation, is yet to be investigated. To understand the disease progression more clearly, we have carried out a systematic study of the pathogen localization by GUS staining of inoculated tomato seedlings, at 24-hour intervals from 0 days post-inoculation (dpi) to 5 dpi. In both inoculation methods, pathogen colonization was observed at 1 dpi at the apical meristem as well as the cotyledon leaves, where the disease initiates. As the disease progressed, colonization by the pathogen towards the lower region of the shoot was observed. Disease consistency and pathogenicity magnitude were observed to be higher using the leaf inoculation method than the root inoculation method. Several R. solanacearum transposon-induced mutants that were reduced in virulence by root inoculation but virulent by leaf inoculation were obtained. Using GUS staining, it was observed that these mutants were unable to localize in the shoot region when inoculated in the root. Our study indicates that the apical meristem and the cotyledon leaves are the first regions to be colonized in inoculated tomato seedlings, which might explain the disease initiation from this region.     

Occurrence of two races of Puccinia kuehnii causing orange rust of sugarcane in Florida

Orange rust of sugarcane caused by Puccinia kuehnii was first reported in Florida in 2007. Since then, several sugarcane cultivars that were resistant during the initial epidemics became susceptible within a few years. These shifts in resistance were attributed to the evolution of the pathogen and appearance of new races. To study the variation in virulence of P. kuehnii, healthy leaf pieces of sugarcane cultivars susceptible to orange rust were brush inoculated with isolates of P. kuehnii collected from susceptible cultivars in the field. After inoculation, leaf pieces were placed in an incubator and disease severity based on the number of rust uredinia was determined 2 weeks postinoculation. Isolates of P. kuehnii collected from sugarcane cultivar CP 89-2143, which only showed severe symptoms of orange rust starting in 2011–2012, produced 300%–500% more uredinia on CP 89-2143 than the isolates collected from cultivar CL 85-1040 that has been susceptible since 2007. Sugarcane cultivar CL 85-1040 exhibited high and equivalent numbers of uredinia regardless of the inoculated isolate of the pathogen. These data support the occurrence of pathogenic specialization within P. kuehnii and the existence of at least two races of this pathogen in Florida. Analysis of amplified fragment-length polymorphism among isolates of P. kuehnii from cultivars CP 89-2143 and CL 85-1040 differing in resistance to orange rust revealed genetic variation among rust uredinia. However, this variation was not associated with a specific sugarcane cultivar, suggesting that pathogenic variation was not linked to major, but rather to small genetic changes within the genome of P. kuehnii.     

燕麦叶斑病病原菌鉴定及其生物学特性

为明确内蒙古、河北地区燕麦叶斑病病原菌种类及其生物学特性,采用组织分离法对病原菌进行分离,通过形态学特征及18SrDNA序列分析进行分类鉴定,以离体和活体叶片接种进行致病性测定,并采用十字交叉法对病原菌的生物学特性进行研究。结果表明,共分离到3株菌株R1、H44和B8,根据形态学特征和18SrDNA序列分析将3株菌株均鉴定为燕麦内脐蠕孢菌Drechslera avenacea(M.A.Curtis ex Cooke)Shoemaker,有性态为燕麦核腔菌Pyrenophora avenae S.Ito et Kurib.。3株菌株均为致病菌,在离体和活体叶片上均能产生腐烂坏死病斑。3株菌株的菌丝在5~30℃、pH 5~11范围内均可生长,其中菌株R1和H44的最适生长温度为25℃、最适生长pH为8,菌株B8的最适生长温度为20~25℃、最适生长pH为7~8。3株菌株对碳源的利用效果中均以淀粉最好,对乳糖利用效果最差;菌株R1的最适氮源是硝酸铵,对蛋白胨和尿素利用效果最差;菌株H44和B8的最适氮源是蛋白胨,菌株H44对硫酸铵利用效果较差,3株菌株都不能利用碳酸铵,且菌株B8也不能利用尿素。     

Note: Comparison of three laboratory methods to evaluate the pathogenicity and virulence of tenPseudomonas syringae pv.syringae strains on apple, pear, cherry and peach trees

The pathogenicity and virulence of ten GreekPseudomonas syringae pv.syringae strains from different hosts (citrus, pear, apple, peach and cherry) were evaluated using three different laboratory methods, which produced results in good agreement. All ten strains were virulent on apple, pear, cherry and peach trees. The extent of tissue colonized varied considerably among strains and cultivars. On excised shoots and twigs of apple and pear, strains BPI 176, BPI 203, PI 2 and PI 14 were the most virulent and strains BPI 689, BPI 992, BPI 4, BPI 20, PI 18 and PI 19 were the least virulent. On excised shoots and twigs of peach and cherry, strains BPI 176, BPI 203, PI 2, PI 14, PI 18 and PI 19 were the most virulent and strains BPI 4 and BPI 20 were the least virulent. Moderate virulence was evinced by strains BPI 689 and BPI 992. These pathogenicity assays are proposed as rapid and reproducible screening systems to evaluate the susceptibility of apple, pear, cherry and peach cultivars to this bacterial pathogen.     

Impact of <Emphasis Type="Italic">Sugarcane yellow leaf virus</Emphasis> on growth and sugar yield of sugarcane

A survey revealed that Sugarcane yellow leaf virus (SCYLV) is found on all Hawaiian sugarcane plantations including those where no yellow leaf symptoms were observed. In a comparison of growth and yield between SCYLV-infected and SCYLV-free plants of the cultivar H87-4094, germination and early shoot growth of infected plants were retarded. The number of stalks per stool was reduced by 30%, biomass was reduced by 29%, and sugar yield by 26% when plants were harvested after 11 months. Yields did not decrease when plants were harvested after 2 years. Thus, SCYLV could reduce yield, even when the plants were asymptomatic. In a field test of SCYLV-susceptible (infected) and -resistant cultivars to compare growth and yield, 10 commercial cultivars (six susceptible and four resistant to SCYLV) were grown in eight fields with different climates and soils. Primary stalk length, biomass and sugar yield did not differ between susceptible and resistant cultivars under any field conditions. Thus, harmful effects of SCYLV on yield cannot be deduced by comparing different cultivars.     

Amplification Polymorphism Among Xanthomonas albilineans Strains, Using a Single Oligonucleotide Primer

A genomic library was produced by a subtractive hybridisation method using DNA from Xanthomonas albilineans serovar I and X. albilineans serovar II, originating from Mauritius. The cloned fragments were amplified and used as probes for Southern hybridisation. Probe F20 was selected on the basis of the RFLP pattern. Upon hybridisation of X. albilineans DNA with probe F20, strains of serovars I and II were differentiated by their banding profiles. This probe was sequenced and oligonucleotide primers were designed. Fragment number and length polymorphisms were obtained after PCR amplification of X. albilineans DNA using F20A as a single primer. The number and size of bands obtained with this primer was correlated to the serotypes of the strains and to the DNA grouping reported by Alvarez et al. (1996). The two serotypes I and II which exist in Mauritius were differentiated by the PCR using primer F20A. The probe and primer developed provide rapid and precise tools for the differentiation of genetic variants within the species X. albilineans.