Roq1 mediates recognition of the Xanthomonas and Pseudomonas effector proteins XopQ and HopQ1

Xanthomonas spp. are phytopathogenic bacteria that can cause disease on a wide variety of plant species resulting in significant impacts on crop yields. Limited genetic resistance is available in most crop species and current control methods are often inadequate, particularly when environmental conditions favor disease. The plant Nicotiana benthamiana has been shown to be resistant to Xanthomonas and Pseudomonas due to an immune response triggered by the bacterial effector proteins XopQ and HopQ1, respectively. We used a reverse genetic screen to identify Recognition of XopQ 1 (Roq1), a nucleotide‐binding leucine‐rich repeat (NLR) protein with a Toll‐like interleukin‐1 receptor (TIR) domain, which mediates XopQ recognition in N. benthamiana. Roq1 orthologs appear to be present only in the Nicotiana genus. Expression of Roq1 was found to be sufficient for XopQ recognition in both the closely‐related Nicotiana sylvestris and the distantly‐related beet plant (Beta vulgaris). Roq1 was found to co‐immunoprecipitate with XopQ, suggesting a physical association between the two proteins. Roq1 is able to recognize XopQ alleles from various Xanthomonas species, as well as HopQ1 from Pseudomonas, demonstrating widespread potential application in protecting crop plants from these pathogens.     

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.     

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     

Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Pest and disease problems are important constraints of cassava production and host plant resistance is the most efficient method of combating them. Breeding for host plant resistance is considerably slowed down by the crop’s biological constraints of a long growth cycle, high levels of heterozygosity and a large genetic load. More efficient methods such as gene cloning and transgenesis are required to deploy resistance genes. To facilitate the cloning of resistance genes, bacterial artificial chromosome (BAC) library resources have been developed for cassava. Two libraries were constructed from the cassava clones, TMS 30001, resistant to the cassava mosaic disease (CMD) and the cassava bacterial blight (CBB), and MECU72, resistant to cassava white fly. The TMS30001 library has 55 296 clones with an insert size range of 40–150 kb with an average of 80 kb, while the MECU72 library consists of 92 160 clones and an insert size range of 25–250 kb average of 93 kb. Based on a genome size of 772 Mb, the TMS30001 and MECU72 libraries have a 5 and 11.3 haploid genome equivalents and a 95 and 99 chance of finding any sequence, respectively. To demonstrate the potential of the libraries, the TMS30001 library was screened by southern hybridization using a cassava analog (CBB1) of the Xa21 gene from rice that maps to a region containing a QTL for resistance to CBB as probe. Five BAC clones that hybridized to CBB1 were isolated and a Hind III fingerprint revealed 2–3 copies of the gene in individual BAC clones. A larger scale analysis of resistance gene analogs (RGAs) in cassava has also been conducted in order to understand the number and organization of RGAs. To scan for gene and repeat DNA content in the libraries, end-sequencing was performed on 2301 clones from the MECU72 library. A total of 1705 unique sequences were obtained with an average size of 715 bp. Database homology searches using BLAST revealed that 458 sequences had significant homology with known proteins and 321 with transposable elements. The use of the library in positional cloning of pest and disease resistance genes is discussed.     

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

Cassava brown streak disease (CBSD) is a leading cause of cassava losses in East and Central Africa, and is currently having a severe impact on food security. The disease is caused by two viruses within the Potyviridae family: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), which both encode atypical Ham1 proteins with highly conserved inosine triphosphate (ITP) pyrophosphohydrolase (ITPase) domains. ITPase proteins are widely encoded by plant, animal, and archaea. They selectively hydrolyse mutagenic nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby function to reduce mutation rates. It has previously been hypothesized that U/CBSVs encode Ham1 proteins with ITPase activity to reduce viral mutation rates during infection. In this study, we investigate the potential roles of U/CBSV Ham1 proteins. We show that both CBSV and UCBSV Ham1 proteins have ITPase activities through in vitro enzyme assays. Deep-sequencing experiments found no evidence of the U/CBSV Ham1 proteins providing mutagenic protection during infections of Nicotiana hosts. Manipulations of the CBSV_Tanza infectious clone were performed, including a Ham1 deletion, ITPase point mutations, and UCBSV Ham1 chimera. Unlike severely necrotic wild-type CBSV_Tanza infections, infections of Nicotiana benthamiana with the manipulated CBSV infectious clones do not develop necrosis, indicating that that the CBSV Ham1 is a necrosis determinant. We propose that the presence of U/CBSV Ham1 proteins with highly conserved ITPase motifs indicates that they serve highly selectable functions during infections of cassava and may represent a euphorbia host adaptation that could be targeted in antiviral strategies.     

The role of the whitefly,Bemisia tabaci (Gennadius), and farmer practices in the spread of cassava brown streak ipomoviruses

Cassava brown streak disease (CBSD) is arguably the most dangerous current threat to cassava, which is Africa's most important food security crop. CBSD is caused by two RNA viruses: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). The roles of the whitefly Bemisia tabaci (Gennadius) and farmer practices in the spread of CBSD were investigated in a set of field and laboratory experiments. The virus was acquired and transmitted by B. tabaci within a short time (5–10 min each for virus acquisition and inoculation), and was retained for up to 48 hr. Highest virus transmission (60%) was achieved using 20–25 suspected viruliferous whiteflies per plant that were given acquisition and inoculation periods of 24 and 48 hr, respectively. Experiments mimicking the agronomic practices of cassava leaf picking or the use of contaminated tools for making cassava stem cuttings did not show the transmission of CBSV or UCBSV. Screenhouse and field experiments in Tanzania showed that the spread of CBSD next to spreader rows was high, and that the rate of spread decreased with increasing distance from the source of inoculum. The disease spread in the field up to a maximum of 17 m in a cropping season. These results collectively confirm that CBSV and UCBSV are transmitted by B. tabaci semipersistently, but for only short distances in the field. This implies that spread over longer distances is due to movements of infected stem cuttings used for planting material. These findings have important implications for developing appropriate management strategies for CBSD.     

Prevalence of Xanthomonas euvesicatoria on Pepper in Brazil

Bacterial spot, caused by Xanthomonas spp., is one of the major diseases of pepper in Brazil, causing considerable losses to crop productivity. The efficient management of the disease is hampered by the high variability of the causal agents. In Brazil, there is no knowledge of which species of Xanthomonas occurs on pepper. In this study, 59 strains of Xanthomonas spp. isolated from different pepper‐producing regions of Brazil were characterized by biochemical and molecular techniques. Results showed the prevalence of X. euvesicatoria as the causal agent of bacterial spot on pepper in Brazil.     

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     

Comparative analysis of genetic variation among <Emphasis Type="Italic">Xanthomonas axonopodis pv manihotis</Emphasis> isolated from the western states of Nigeria using RAPD and AFLP

Xanthomonas axonopodis pv manihotis is the causal agent of cassava bacterial blight (CBB) worldwide. CBB disease is a major constraint to cassava cultivation, and losses can be extremely severe in regions where highly susceptible cultivars are grown. To develop an efficient disease management policy, the genetic diversity of the pathogens population must be known. There is dearth of information on the genetic diversity of X. axonopodis pv manihotis population in Nigeria. We used RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism), a PCR-based technique, to characterize the X. axonopodis pv manihotis isolates from the western States of Nigeria. Thirteen strains Xam and 2 reference strains were tested with eight primers combination of AFLP and 4 RAPD primers. RAPD amplified DNA fragment data showed four major clusters at 80 % similarity coefficient level and two strains were not clustered by this analysis. Strains Kwa76A and Ond48A were also separated in the principal component analysis of the same data. Numerical analysis differentiated the AFLP patterns into four distinct clusters and grouped two strains separately at 66 % similarity. PCA assembly grouped the bacterial strains into 4 and one of the strains was singled out from the others. The two DNA analyses techniques seem to be complimentary to one another and informative on the genomic structure of Xam population in Western Nigeria. The genetic analysis presented here contributes to understanding of the Xam population structure in Western Nigeria.     

Virulence and type III effector diversities of Xanthomonas citri pv. fuscans and X. phaseoli pv. phaseoli in Brazil

Common bacterial blight (CBB) is caused by four genetic lineages belonging to two species of Xanthomonas, namely Xanthomonas citri pv. fuscans (includes fuscans, NF2 and NF3 lineages) and X. phaseoli pv. phaseoli (lineage NF1). A collection of 117 strains of Xanthomonas isolated from common bean plants grown in several producing regions of Brazil, between 2007 and 2016 was established. For species and lineage identification, the following tests were performed: multiplex PCR with a set of four specific primer pairs, pathogenicity tests on susceptible cultivar BRS Artico and phylogenetic analysis based on housekeeping gene sequences. The presence of the two species were confirmed among the 117 strains, being 62 non-fuscans strains (NF1, NF2 and NF3) and 55 fuscans strains of X. citri pv. fuscans. To select a set of representative strains for the virulence assay, a PCR-based analysis of effector diversity was performed with 42 strains belonging to the two species. PCR with primers for xopL, avrBsT, xopE2 and xopE1 genes were positive for all strains, while for the other six effectors there was variation. Six distinct effector profiles were detected, and one strain representing each type was inoculated in 15 common bean cultivars with varying levels of resistance to CBB. The fuscans strains showed uniformity in their effector profiles and were the most virulent. The phylogenetic analyses of our strain collection revealed that all genetic variants of CBB pathogens (NF1, NF2, NF3 and fuscans) are present in Brazil, with significant variability in virulence to common bean cultivars.     

Autophagy-related genes serve as heat shock protein 90 co-chaperones in disease resistance against cassava bacterial blight

Heat shock protein 90 (HSP90) is involved in plant growth and various stress responses via regulating protein homeostasis. Autophagy keeps cellular homeostasis by recycling the components of cellular cytoplasmic constituents. Although they have similar effects on cellular protein homeostasis, the direct association between HSP90 and autophagy signaling remains unclear in plants, especially in tropical crops. In this study, the correlation between HSP90 and autophagy signaling was systematically analyzed by protein–protein interaction in cassava, one of the most important economy fruit in tropic. In addition, their effects on plant disease response and underlying mechanisms in cassava were investigated by functional genomics and genetic phenotype assay. The potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex interacts with MeATGs and subsequently triggers autophagy signaling, conferring improved disease resistance to cassava bacterial blight (CBB). On the contrary, HSP90 inhibitor and autophagy inhibitor decreased disease resistance against CBB in cassava, and autophagy may be involved in the potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex-mediated multiple immune responses. This study highlights the precise modulation of autophagy signaling by potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex in autophagy-mediated disease resistance to CBB.     

The Status of Cassava Bacterial Blight Caused by Xanthomonas campestris pv. manihotis in Trinidad

Disease surveys conducted in Trinidad between 1985—1987 showed that Cassava Bacterial Blight (CBB) is present in all but one county of the country with disease severity ratings varying from 1—5 depending on day/night temperatures. Field and greenhouse screening identified varieties such as Point Fortin fine leaf and CMC 40 as being resistant whereas M col 22 was moderately resistant to susceptible. Using a combination of antiserum produced to whole cells of Xanthomonas campestris pv. manibotis and a broth enrichment technique, dissemination of the pathogen by flood water was confirmed. The pathogen was detected at distances of up to 300 meters from infected fields. The significance of this mode of pathogen dissemination in initiating primary infection in Trinidad is discussed.     

An EST resource for cassava and other species of Euphorbiaceae

Cassava (Manihot esculenta) is a major food staple for nearly 600 million people in Africa, Asia, and Latin America. Major losses in yield result from biotic and abiotic stresses that include diseases such as Cassava Mosaic Disease (CMD) and Cassava Bacterial Blight (CBB), drought, and acid soils. Additional losses also occur from deterioration during the post-harvest storage of roots. To help cassava breeders overcome these obstacles, the scientific community has turned to modern genomics approaches to identify key genetic characteristics associated with resistance to these yield-limiting factors. One approach for developing a genomics program requires the development of ESTs (expressed sequence tags). To date, nearly 23000 ESTs have been developed from various cassava tissues, and genotypes. Preliminary analysis indicates existing EST resources contain at least 6000–7000 unigenes. Data presented in this report indicate that the cassava ESTs will be a valuable resource for the study of genetic diversity, stress resistance, and growth and development, not only in cassava, but also other members of the Euphorbiaceae family.     

Cassava mosaic geminiviruses in Africa

Cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses (CMGs) (Geminiviridae:Begomovirus) is undoubtedly the most important constraint to the production of cassava in Africa at the outset of the 21st century. Although the disease was recorded for the first time in the latter part of the 19th century, for much of the intervening period it has been relatively benign in most of the areas where it occurs and has generally been considered to be of minor economic significance. Towards the end of the 20th century, however, the inherent dynamism of the causal viruses was demonstrated, as a recombinant hybrid of the two principal species was identified, initially from Uganda, and shown to be associated with an unusually severe and rapidly spreading epidemic of CMD. Subsequent spread throughout East and Central Africa, the consequent devastation of production of the cassava crop, a key staple in much of this region, and the observation of similar recombination events elsewhere, has once again demonstrated the inherent danger posed to man by the capacity of these viruses to adapt to their environment and optimally exploit their relationships with the whitefly vector, plant host and human cultivator. In this review of cassava mosaic geminiviruses in Africa, we examine each of these relationships, and highlight the ways in which the CMGs have exploited them to their own advantage.     

Bacterial Diseases on Witloof Chicory Crops in Europe

Distribution of principal phytopathogenic bacterial populations on witloof chicory crops in Europe is reported. Different symptoms caused by phytopathogenic bacterial populations on different parts of witloof chicory plants are described. The first report of Pseudomonas fluorescens (Trevisan) Migula and occurrence of Erwinia carotovora (Jones, 1901) bacterial populations on witloof chicory cv. Rosso di Chioggia crops in Italy (Latium region), are reported. Risks of disease during different phases of production of this crop and phytosanitary measures to reduce the incidence of disease caused by these pathogens are discussed.     

Sensitive Detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by Loop-Mediated Isothermal Amplification

Molecular diagnostics for crop diseases can enhance food security by enabling the rapid identification of threatening pathogens and providing critical information for the deployment of disease management strategies. Loop-mediated isothermal amplification (LAMP) is a PCR-based tool that allows the rapid, highly specific amplification of target DNA sequences at a single temperature and is thus ideal for field-level diagnosis of plant diseases. We developed primers highly specific for two globally important rice pathogens, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) disease, and X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak disease (BLS), for use in reliable, sensitive LAMP assays. In addition to pathovar distinction, two assays that differentiate X. oryzae pv. oryzae by African or Asian lineage were developed. Using these LAMP primer sets, the presence of each pathogen was detected from DNA and bacterial cells, as well as leaf and seed samples. Thresholds of detection for all assays were consistently 10⁴ to 10⁵ CFU ml⁻¹, while genomic DNA thresholds were between 1 pg and 10 fg. Use of the unique sequences combined with the LAMP assay provides a sensitive, accurate, rapid, simple, and inexpensive protocol to detect both BB and BLS pathogens.