The tomato I gene for Fusarium wilt resistance encodes an atypical leucine‐rich repeat receptor‐like protein whose function is nevertheless dependent on SOBIR1 and SERK3/BAK1

We have identified the tomato I gene for resistance to the Fusarium wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol) and show that it encodes a membrane‐anchored leucine‐rich repeat receptor‐like protein (LRR‐RLP). Unlike most other LRR‐RLP genes involved in plant defence, the I gene is not a member of a gene cluster and contains introns in its coding sequence. The I gene encodes a loopout domain larger than those in most other LRR‐RLPs, with a distinct composition rich in serine and threonine residues. The I protein also lacks a basic cytosolic domain. Instead, this domain is rich in aromatic residues that could form a second transmembrane domain. The I protein recognises the Fol Avr1 effector protein, but, unlike many other LRR‐RLPs, recognition specificity is determined in the C‐terminal half of the protein by polymorphic amino acid residues in the LRRs just preceding the loopout domain and in the loopout domain itself. Despite these differences, we show that I/Avr1‐dependent necrosis in Nicotiana benthamiana depends on the LRR receptor‐like kinases (RLKs) SERK3/BAK1 and SOBIR1. Sequence comparisons revealed that the I protein and other LRR‐RLPs involved in plant defence all carry residues in their last LRR and C‐terminal LRR capping domain that are conserved with SERK3/BAK1‐interacting residues in the same relative positions in the LRR‐RLKs BRI1 and PSKR1. Tyrosine mutations of two of these conserved residues, Q922 and T925, abolished I/Avr1‐dependent necrosis in N. benthamiana, consistent with similar mutations in BRI1 and PSKR1 preventing their interaction with SERK3/BAK1.     

Investigation of the diversity of effector genes in the banana pathogen,Fusarium oxysporum f. sp. cubense,reveals evidence of horizontal gene transfer

It is hypothesized that the virulence of phytopathogenic fungi is mediated through the secretion of small effector proteins that interfere with the defence responses of the host plant. In Fusarium oxysporum, one family of effectors, the Secreted In Xylem (SIX) genes, has been identified. We sought to characterize the diversity and evolution of the SIX genes in the banana‐infecting lineages of F. oxysporum f. sp. cubense (Foc). Whole‐genome sequencing data were generated for the 23 genetic lineages of Foc, which were subsequently queried for the 14 known SIX genes (SIX1–SIX14). The sequences of the identified SIX genes were confirmed in a larger collection of Foc isolates. Genealogies were generated for each of the SIX genes identified in Foc to further investigate the evolution of the SIX genes in Foc. Within Foc, variation of the SIX gene profile, including the presence of specific SIX homologues, correlated with the pathogenic race structure of Foc. Furthermore, the topologies of the SIX gene trees were discordant with the topology of an infraspecies phylogeny inferred from EF‐1α/RPB1/RPB2 (translation elongation factor‐1α/RNA polymerase II subunit I/RNA polymerase II subunit II). A series of topological constraint models provided strong evidence for the horizontal transmission of SIX genes in Foc. The horizontal inheritance of pathogenicity genes in Foc counters previous assumptions that convergent evolution has driven the polyphyletic phylogeny of Foc. This work has significant implications for the management of Foc, including the improvement of diagnostics and breeding programmes.     

Identification of pathogenicity‐related genes in Fusarium oxysporum f. sp. cepae

Pathogenic isolates of Fusarium oxysporum, distinguished as formae speciales (f. spp.) on the basis of their host specificity, cause crown rots, root rots and vascular wilts on many important crops worldwide. Fusarium oxysporum f. sp. cepae (FOC) is particularly problematic to onion growers worldwide and is increasing in prevalence in the UK. We characterized 31 F. oxysporum isolates collected from UK onions using pathogenicity tests, sequencing of housekeeping genes and identification of effectors. In onion seedling and bulb tests, 21 isolates were pathogenic and 10 were non‐pathogenic. The molecular characterization of these isolates, and 21 additional isolates comprising other f. spp. and different Fusarium species, was carried out by sequencing three housekeeping genes. A concatenated tree separated the F. oxysporum isolates into six clades, but did not distinguish between pathogenic and non‐pathogenic isolates. Ten putative effectors were identified within FOC, including seven Secreted In Xylem (SIX) genes first reported in F. oxysporum f. sp. lycopersici. Two highly homologous proteins with signal peptides and RxLR motifs (CRX1/CRX2) and a gene with no previously characterized domains (C5) were also identified. The presence/absence of nine of these genes was strongly related to pathogenicity against onion and all were shown to be expressed in planta. Different SIX gene complements were identified in other f. spp., but none were identified in three other Fusarium species from onion. Although the FOC SIX genes had a high level of homology with other f. spp., there were clear differences in sequences which were unique to FOC, whereas CRX1 and C5 genes appear to be largely FOC specific.     

Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes

The tomato I‐3 and I‐7 genes confer resistance to Fusarium oxysporum f. sp. lycopersici (Fol) race 3 and were introgressed into the cultivated tomato, Solanum lycopersicum, from the wild relative Solanum pennellii. I‐3 has been identified previously on chromosome 7 and encodes an S‐receptor‐like kinase, but little is known about I‐7. Molecular markers have been developed for the marker‐assisted breeding of I‐3, but none are available for I‐7. We used an RNA‐seq and single nucleotide polymorphism (SNP) analysis approach to map I‐7 to a small introgression of S. pennellii DNA (c. 210 kb) on chromosome 8, and identified I‐7 as a gene encoding a leucine‐rich repeat receptor‐like protein (LRR‐RLP), thereby expanding the repertoire of resistance protein classes conferring resistance to Fol. Using an eds1 mutant of tomato, we showed that I‐7, like many other LRR‐RLPs conferring pathogen resistance in tomato, is EDS1 (Enhanced Disease Susceptibility 1) dependent. Using transgenic tomato plants carrying only the I‐7 gene for Fol resistance, we found that I‐7 also confers resistance to Fol races 1 and 2. Given that Fol race 1 carries Avr1, resistance to Fol race 1 indicates that I‐7‐mediated resistance, unlike I‐2‐ or I‐3‐mediated resistance, is not suppressed by Avr1. This suggests that Avr1 is not a general suppressor of Fol resistance in tomato, leading us to hypothesize that Avr1 may be acting against an EDS1‐independent pathway for resistance activation. The identification of I‐7 has allowed us to develop molecular markers for marker‐assisted breeding of both genes currently known to confer Fol race 3 resistance (I‐3 and I‐7). Given that I‐7‐mediated resistance is not suppressed by Avr1, I‐7 may be a useful addition to I‐3 in the tomato breeder's toolbox.     

Biocontrol of Fusarium Wilt and Growth Promotion of Tomato Plants Using Endophytic Bacteria Isolated from Solanum elaeagnifolium Stems

Seven culturable bacterial isolates, obtained from the internal stem tissues of Solanum elaeagnifolium and successfully colonizing the internal stem tissues of tomato cv. Rio Grande, were screened for their in vivo antifungal activity against Fusarium oxysporum f.sp. lycopersici (FOL) and their growth‐promoting potential on tomato plants. SV101 and SV104 isolates, assessed on pathogen‐challenged tomato plants led to a significant decrease (77–83%) in Fusarium wilt severity and vascular browning extent (76%), as compared to the inoculated and untreated control. Isolates enhanced growth parameters on pathogen‐challenged and unchallenged tomato plants. SV104 and SV101 isolates were most effective in suppressing disease and enhancing plant growth. These two isolates were identified as Bacillus sp. str. SV101 ( KU043040 ) and B. tequilensis str. SV104 ( KU976970 ). They displayed antifungal activity against FOL; pathogen growth was inhibited by 64% and an inhibition zone (11.50 and 19.75 mm) against FOL could be formed using whole cell suspensions. SV101 and SV104 extracellular metabolites also inhibited FOL growth by 20 and 55%, respectively, as compared to control. B. tequilensis str. SV104 was shown to produce protease, chitinase, pectinase, IAA and siderophores. Bacillus sp. str. SV101 displayed pectinase activity and was found to be an IAA‐producing and phosphate‐solubilizing agent. To our knowledge, this is the first study reporting on S. elaeagnifolium use as a potential source of potent biocontrol and plant growth‐promoting agents.     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

A putative endophytic Bacillus cereus str. S42 from Nicotiana glauca for biocontrol of Fusarium wilt disease in tomato and gas chromatography-mass spectrometry analysis of its chloroform extract

A putative endophytic Bacillus cereus str. S42 (KP993206), recovered from surface-sterilised stems of Nicotiana glauca was assessed in vitro and in vivo for its antifungal potential towards Fusarium oxysporum f. sp. lycopersici (FOL). Pathogen sporulation was significantly inhibited by B. cereus str. S42. FOL mycelial growth was reduced using its whole-cell suspensions, cell-free culture supernatant and chloroform extract. Its extracellular metabolites remained effective after heating at 50–100 °C with a decline in their activity was observed beyond 100 °C, when added with proteinase K and/or after pH adjustment to 2 and 12. Chitinase gene was detected using PCR amplification. Gas chromatography–mass spectrometry analysis of its chloroform extract matched phthalic acid, dibutyl ester with high level of similarity. B. cereus str. S42 cell-free culture supernatant and whole-cell suspensions had significantly suppressed Fusarium wilt severity by 87–96% and enhanced tomato growth by 39–79% compared to FOL-inoculated and untreated control.     

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. III. PR-protein gene expression and ethylene evolution in tomato cell lines transgenic for phytohormone-related bacterial genes

 Previous work carried out in our laboratory has shown that, in tomato, the alteration of endogenous phytohormone equilibria through the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis can modify the active defense response to Fusarium oxysporum f. sp. lycopersici. The susceptible cv ‘Red River’ acquires a stable competence for active defense, particularly when the phytohormone equilibrium is altered in favour of cytokinins. Here, we analyse the expression of genes involved in the defense response against pathogens, i.e. pathogenesis-related (PR)-protein genes, in the susceptible ‘Red River’ and resistant ‘Davis’ cultivars transgenic for the aforementioned genes. Fungal cell-wall components, glutathione, salicylic acid and the ethylene-forming ethephon are used as “probes” for the induction of defense processes, including ethylene production. The data obtained show that the extracellular PR-proteins (acidic chitinase and PR-1 protein) that were inducible in the control tissue of the resistant ‘Davis’ cultivar and not expressed in the susceptible ‘Red River’ cultivar became constitutive in the transgenic tissues of both. On the other hand, expression of the intracellular PR-proteins (basic chitinase and β-1,3-glucanase) was found to be constitutive in all cases, both in the control and in the transgenic cell lines of the resistant and the susceptible tomato cultivars. Ethylene production was higher in ‘Davis’ than in ‘Red River’, and significantly increased in the transgenic cell lines, particularly when cytokinin synthesis was altered. Received: 25 February 1998 / Accepted: 7 April 1998