A Complex Population Structure of the Cassava Pathogen Xanthomonas axonopodis pv. manihotis in Recent Years in the Caribbean Region of Colombia

Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is the most important bacterial disease affecting this crop. A continuous surveillance of the pathogen population dynamics is required to develop an efficient disease management program. During the 1990s, Xam populations showed high levels of genetic variation and relevant migratory processes that were important determinants of the distribution of the pathogen diversity in Colombia. Aiming to characterize the current population structure of the pathogen and the evolutionary forces that shape these populations, sampling collections were carried out from September 2008 until November 2010 in the Colombian Caribbean Region. One hundred and sixty bacterial isolates were characterized using amplified fragment length polymorphism (AFLP) markers. Additionally, a subset of effector genes were sequenced in some isolates to determine their usefulness in Xam population studies and to provide additional information to that obtained with AFLPs. Virulence patterns of ten isolates were determined in nine cassava accessions. Our results show a complex architecture of population and confirm migratory process previously reported in the Caribbean Region. Chinú, one of the locations sampled, presented remarkable features in population dynamics such as longer genetic distances, higher diversity indices, and a genetically differentiated population when it was compared with other locations. Virulence tests showed that MCOL2215, one of the most cultivated cassava varieties in the Caribbean coast, was susceptible to the majority of Xam isolates tested. This study shows the current condition of populations of Xam in the Caribbean Region of Colombia, and it contributes to improve the existing bacterial blight control practices.     

Genetic mapping of resistance to bacterial blight disease in cassava (Manihot esculenta Crantz)

Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is a major disease of cassava (Manihot esculenta Crantz) in Africa and South America. Planting resistant varieties is the preferred method of disease control. Recent genetic mapping of an F₁ cross (TMS 30572 × CM 2177–2) led to the construction of the first molecular genetic map of cassava. To better understand the genetics of resistance to CBB, we evaluated individuals of the F₁ cross for CBB resistance by controlled greenhouse inoculations and visually assessed symptoms on days 7, 15, and 30 days after inoculation, using a scale where 0 = no disease and 5 = maximum susceptibility. Five Xam strains were used: CIO-84, CIO-1, CIO-136, CIO-295, and ORST X-27. Area under the disease progress curve (AUDPC) was used as a quantitative measure of resistance in QTL analysis by single-marker regression. Based on the AUDPC values, eight QTLs (quantitative trait loci), located on linkage groups B, D, L, N, and X of the female-derived framework map, were found to explain 9–20% of the phenotypic variance of the crop’s response to the five Xam strains. With the male-derived framework map, four QTLs on linkage groups G and C explained 10.7–27.1% of the variance. A scheme to confirm the usefulness of these markers in evaluating segregating populations for resistance to CBB is proposed. Received: 20 September 1999 / Accepted: 30 December 1999     

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.     

The role of type III effectors from Xanthomonas axonopodis pv. manihotis in virulence and suppression of plant immunity

Xanthomonas axonopodis pv. manihotis (Xam) causes cassava bacterial blight, the most important bacterial disease of cassava. Xam, like other Xanthomonas species, requires type III effectors (T3Es) for maximal virulence. Xam strain CIO151 possesses 17 predicted T3Es belonging to the Xanthomonas outer protein (Xop) class. This work aimed to characterize nine Xop effectors present in Xam CIO151 for their role in virulence and modulation of plant immunity. Our findings demonstrate the importance of XopZ, XopX, XopAO1 and AvrBs2 for full virulence, as well as a redundant function in virulence between XopN and XopQ in susceptible cassava plants. We tested their role in pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) and effector‐triggered immunity (ETI) using heterologous systems. AvrBs2, XopR and XopAO1 are capable of suppressing PTI. ETI suppression activity was only detected for XopE4 and XopAO1. These results demonstrate the overall importance and diversity in functions of major virulence effectors AvrBs2 and XopAO1 in Xam during cassava infection.     

Genomic Survey of Pathogenicity Determinants and VNTR Markers in the Cassava Bacterial Pathogen Xanthomonas axonopodis pv. Manihotis Strain CIO151

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease.     

Gene expression profile in response to <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">manihotis</Emphasis> infection in cassava using a cDNA microarray

A cassava cDNA microarray based on a large cassava EST database was constructed and used to study the incompatible interaction between cassava and Xanthomonas axonopodis pv. manihotis (Xam) strain CIO151. For microarray construction, 5700 clones from the cassava unigene set were amplified by polymerase chain reaction (PCR) and printed on glass slides. Microarray hybridization was performed using cDNA from cassava plants (resistant variety MBra685) collected at 12, 24, 48 h and 7 and 15 days post-infection as treatment and cDNA from mock-inoculated plants as control. A total of 199 genes were found to be differentially expressed (126 up-regulated and 73 down-regulated). A greater proportion of differentially-expressed genes was observed at 7 days after inoculation. Expression profiling and cluster analyses indicate that, in response to inoculation with Xam, cassava induces dozens of genes, including principally those involved in oxidative burst, protein degradation and pathogenesis-related (PR) genes. In contrast, genes encoding proteins that are involved in photosynthesis and metabolism were down regulated. In addition, various other genes encoding proteins with unknown function or showing no similarity to other proteins were also induced. Quantitative real time PCR experiments confirmed the reliability of our microarray data. In addition we showed that some genes are induced more rapidly in the resistant than in the susceptible cultivar.These authors made equal contributions to this work.     

QTL analysis of field resistance to Xanthomonas axonopodis pv. manihotis in cassava

We evaluated cassava bacterial blight (CBB) infection in an pair-cross population of 150 individuals derived from an intra-specific cross between two non-inbred cassava (Manihot esculenta Crantz) lines. The replicated trials were carried out in the field under high disease pressure over two consecutive crop cycles. Evaluations were conducted at 4 and 7 months after planting for the two cycles. Simple regression analysis and the nonparametric Kruskal-Wallis rank-sum test revealed that eight quantitative trait loci (QTLs) were involved in resistance. We detected changes in QTLs from crop cycle to crop cycle. The pathogen population (Xanthomonas axonopodis pv. manihotis) was also monitored over the period, using a restriction fragment length polymorphism probe and pathogenic tests. Changes in QTL detection over the 2 years could be correlated with changes in pathogen population structure. One QTL, located in linkage group D, was conserved over the two crop cycles, and in field to greenhouse evaluations. This study thus identified molecular markers useful for marker assisted-selection, a technique that can accelerate the long, multiple-season process of breeding for CBB resistance. Received: 1 January 2000 / Accepted: 25 June 2000     

Genotypic Characterization of the Common Bean Bacterial Blight Pathogens, Xanthomonas axonopodis pv. phaseoli and Xanthomonas axonopodis pv. phaseoli var. fuscans by rep-PCR and PCR–RFLP of the Ribosomal Genes

Common bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli and X. axonopodis pv. phaseoli var. fuscans is one of the most destructive diseases of common bean worldwide. The interrelatedness, genetic diversity and geographical distribution of the CBB pathogens was assessed using restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction amplified 16S ribosomal gene, including the 16S–23S intergenic spacer region and repetitive element PCR (rep‐PCR). RFLP profiles generated by the restriction endonucleases MboI, RsaI and HaeIII differentiated X. axonopodis pv. phaseoli from X. axonopodis pv. phaseoli var. fuscans and non‐pathogenic Xanthomonas species associated with common bean. Cluster analysis of rep‐PCR profiles revealed a high level of genetic differentiation (G_ST = 0.56) between the two CBB pathogens, showing that they are genetically distinct. Significant levels of genetic diversity were observed within each strain, indicating that the two bacteria are not clonal. More genetic diversity was observed in X. axonopodis pv. phaseoli (H = 0.134; I = 0.223) than X. axonopodis pv. phaseoli var. fuscans (H = 0.108; I = 0.184). However, no geographical differentiation was evident for either X. axonopodis pv. phaseoli var. fuscans (GST = 0.013) or X. axonopodis pv. phaseoli (G_ST = 0.017). This lack of geographical differentiation has important practical implications, as available host resistance genes are likely to be effective in controlling the disease in diverse geographical areas.     

Molecular Typing and Genetic Characterization of Pathogenic Variants of Xanthomonas axonopodis pv. citri in Taiwan

Molecular typing was applied and optimized for genetic characterization for three pathogenic variants of Xanthomonas axonopodis pv. citri (Xac) from Taiwan. These three novel variants of atypical symptom–producing X. axonopodis pv. citri were designated as Xac‐A^f, Xac‐A^p and Xac‐A^r. Based on polymerase chain reaction (PCR) with primers specific to X. axonopodis pv. citri, leucine‐responsive regulatory protein (lrp) gene assay and DNA fingerprintings generated by repetitive‐sequence PCR (rep‐PCR) and amplified fragment length polymorphism (AFLP) were used to compare strains including the three types of atypical symptom–producing strains Xac‐A^f, Xac‐A^p and Xac‐A^r, and additional reference strains from pathotypes Xac‐A, Xac‐A*, Xac‐A^w, X. axonopodis pv. auruantifolii and X. axonopodis pv. citrumelo. These three types of X. axonopodis pv. citri variants can be detected with six sets of primer specific for X. axonopodis pv. citri. Cluster analyses by lrp sequence assay, AFLP and combing the band patterns of rep‐PCR clearly grouped the atypical symptom–producing variants in types Xac‐ A^f, Xac‐A^r and Xac‐A^p into the same cluster with typical symptom‐producing strains in pathotype Xac‐A. These three types of X. axonopodis pv. citri variants could be excluded from strains of Xac‐A* and Xac‐A^w in these genotypic analyses. Strains of Xac‐A* and Xac‐A^w were closely related to Xac‐A strains in our results. No Taiwan isolate was related to X. axonopodis pv. auruantifolii or X. axonopodis pv. citrumelo. The results further confirmed the atypical symptom–producing variants of X. axonopodis pv. citri in Taiwan belong to pathotype Xac‐A.     

Cassava Bacterial Blight: Using Genomics for the Elucidation and Management of an Old Problem

Bacterial Blight is an important disease of cassava, causing losses that have resulted in historical famines in certain growing zones. The disease is caused by Xanthomonas axonopodis pv. manihotis, a gram-negative rod that belongs to the gammaproteobacteria. In this review, we describe the pathosystem and the recent studies that have been undertaken to elucidate both susceptibility and resistance mechanisms in cassava, with the hope of generating resistant plants using biotechnology. We first describe studies of the pathogen, including pathogen population changes through time as well as genomic tools that have recently been generated to determine pathogenicity factors. Secondly, we discuss mechanisms of disease resistance that have been elucidated in recent years and how these mechanisms could be used for the generation of improved plants resistant to CBB.     

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.     

Comparison of Randomly Amplified Polymorphic DNA with Amplified Fragment Length Polymorphism To Assess Genetic Diversity and Genetic Relatedness within Genospecies III of Pseudomonas syringae

Recently, DNA pairing analyses showed that Pseudomonas syringae pv. tomato and related pathovars, including P. syringae pv. maculicola, form a genomic species (Pseudomonas tomato) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445-448, in K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed., Pseudomonas syringae Pathovars and Related Pathogens, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of P. syringae cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of P. syringae strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of P. syringae pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other P. syringae pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of P. syringae pv. tomato and P. syringae pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of P. syringae pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique.     

AFLP assessment of genetic variability in cassava accessions (Manihot esculenta) resistant and susceptible to the cassava bacterial blight (CBB).

Cassava bacterial blight (CBB) is caused by Xanthomonas axonopodis pv. manihotis (Xam). Resistance is found in Manihot esculenta and, in addition, has been introgressed from a wild relative, M. glaziovii. The resistance is thought to be polygenic and additively inherited. Ninety-three varieties of M. esculenta (Crantz) were assessed by AFLPs for genetic diversity and for resistance to CBB. AFLP analysis was performed using two primer combinations and a 79.2% level of polymorphism was found. The phenogram obtained showed between 74% and 96% genetic similarity among all cassava accessions analysed. The analysis permitted the unique identification of each individual. Two Xam strains were used for resistance screening. Variation in the reaction of cassava varieties to Xam strains was observed for all plant accessions. The correlation of resistance to both strains, had a coefficient of 0.53, suggesting the independence of resistance to each strain. Multiple correspondence analysis showed a random distribution of the resistance/susceptibility response with respect to overall genetic diversity as measured by AFLP analysis. A total heterozygosity index was calculated to determine the diversity within clusters as well as among them. Our results demonstrate that resistance to CBB is broadly distributed in cassava germplasm and that AFLP analysis is an effective and efficient means of providing quantitative estimates of genetic similarities among cassava accessions.     

Genetic variation in cultivated strains of <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Genetic differences among Agaricus blazei strains were investigated using somatic incompatibility testing, isozyme analysis, restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA), and random amplified polymorphic DNA (RAPD) analysis. Eight strains, one cultivated strain from Brazil and seven from Japan, were used in this study. Somatic incompatibility interactions were observed between the Brazilian cultivated strain and the Japanese strains. The Brazilian cultivated strain had its own distinct patterns of esterase isozyme and mtDNA RFLP, but all seven Japanese cultivated strains showed identical patterns. When the RAPD patterns, obtained using eight primers, were compared the eight strains had their own distinct RAPD profiles. Distance values were calculated between all pairs of the strains based on presence or absence of individual RAPD bands, and a dendrogram was constructed by unweighted pair-group method with arithmetic clustering (UPGMA) analysis. Seven Japanese cultivated strains were grouped to each other, and this group was finally linked to the Brazilian cultivated strain. Based on these results, the degree of genetic variation among the A. blazei strains used is discussed.     

Molecular characterization of intra-population variability of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. using DNA based molecular markers

Jatropha curcas L. (Euphorbiaceae) has acquired a great importance as a renewable source of energy with a number of environmental benefits. Very few attempts were made to understand the extent of genetic diversity of J. curcas germplasm. In the present study, efforts were made to analyze the genetic diversity among the elite germplasms of J. curcas, selected on the basis of their performance in field using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR). The plants were selected on the basis of height, canopy circumference, number of seeds per fruit, weight of 100 seeds, seed yield in grams per plant and oil content. Out of 250 RAPD (with 26 primers), 822 AFLP (with 17 primers) and 19 SSR band classes, 141, 346 and 7 were found to be polymorphic, respectively. The percentage polymorphism among the selected germplasms using RAPD, AFLP and SSR was found to be 56.43, 57.9, and 36.84, respectively. The Jaccard’s similarity coefficient was found 0.91, 0.90 and 0.91 through RAPD, AFLP and SSR marker systems, respectively. Principle component analysis (PCA) and dendrogarm analysis of genetic relationship among the germplasm using RAPD, AFLP and SSR data showed a good correlation for individual markers. The germplasm JCC-11, 12, 13, 14 and 15 whose yield found to be high were clustered together in dendrogram and PCA analysis though JCC11 is geographically distinct from others. In overall analysis JCC6 (in RAPD), JCC8 (in AFLP) and JCC 6 and JCC10 (in SSR) were found genetically diverse. Characterization of geographically distinct and genetically diverse germplasms with varied yield characters is an important step in marker assisted selection (MAS) and it can be useful for breeding programs and QTL mapping.     

GENETIC VARIATION IN ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE): IS IT LINKED TO FREQUENT EPIDEMICS OF HOST‐SPECIFIC PARASITIC FUNGI?1

Understanding of the genetic basis for susceptibility and resistance is still lacking for most aquatic host–parasite systems, for instance, for phytoplankton and their fungal parasites. Fungal parasites can have significant effects on phytoplankton populations, mainly through their ability to decimate algal host populations during epidemics. We used random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analysis to study levels of genetic variation within a population of the freshwater diatom Asterionella formosa Hassall in relation to parasitism by the obligate, host‐specific, fungal parasite Zygorhizidium planktonicum Canter. The level of genetic variation within the A. formosa population in Lake Maarsseveen, The Netherlands was found to be high despite the presumed absence or very low frequency of sexual reproduction in this species, the limited gene flow, and the severity of parasite attack that would purge the population from susceptible genotypes. RAPD analysis revealed four distinct banding patterns, with 3 of 21 markers (14%) being polymorphic. In AFLP analysis, every single isolate of A. formosa showed a unique banding pattern, and 120 of the 210 AFLP markers (57%) were found to be polymorphic. Furthermore, character compatibility analysis revealed that sexual reproduction may be one of the mechanisms that generates and maintains genetic variation in the A. formosa population in Lake Maarsseveen. The presence of genetic variation in A. formosa was reflected in infection experiments, which showed that genetically different A. formosa strains differed in their susceptibility to various Z. planktonicum strains and that parasite strains differed in their ability to infect particular host strains.