Further identification of races of Cladosporium fulvum (Fulvia fulva) on tomato originating from the Netherlands France and Poland

Races ofCladosporium fulvum, which can overcome the resistance of the genes Cf₂, Cf₄, Cf₅, Cf₈, Cf₉ and Cf₁₁ have appeared in the Netherlands, France and Poland. Known isolates from the Netherlands and France and three new isolates from Poland have been investigated for the presence of virulence genes using a set of genotypes carrying resistance genes Cf₂ to Cf₁₁.Several Dutch isolates of races, earlier designated as 2.4, 2.4.5 and 2.4.5.9, were found to break down the resistance gene Cf₁₁. These races must therefore be designated as 2.4.11, 2.4.5.11 and 2.4.5.9.11 respectively. In the new Polish isolates virulence genes, overcoming the resistance genes Cf₂, Cf₄, Cf₈, Cf₉ and Cf₁₁ were found. Since all races able to grow on genotypes with Cf₄, could also grow on genotypes carrying Cf₈, it was impossible to discriminate between the genes Cf₄ and Cf₈. These Polish isolates were designated as races 4.11, 2.4.11 and 2.4.9.11. The consequences of the occurrence of these races for tomato breeding are discussed.Samenvatting Fysio's vanCladosporium fulvum, die de resistentie-genen Cf₂, Cf₄, Cf₅, Cf₈, Cf₉ en Cf₁₁ kunnen doorbreken, zijn in Nederland, Frankrijk en Polen opgetreden. Met behulp van een groep genotypen, die de resistentie genen Cf₂ tot en met Cf₁₁ dragen, zijn Nederlandse, Franse en enkele nieuwe Poolse isolaten onderzocht op de aanwezigheid van virulentiegenen. Enkele Nederlandse isolaten, eerder aangeduid met 2.4, 2.4.5. en 2.4.5.9, bleken het resistentie-gen Cf₁₁ te kunnen doorbreken. Deze moeten daarom aangeduid worden als respectievelijk 2.4.11, 2.4.5.11 en 2.4.5.9.11. In de nieuwe Poolse isolaten werd virulentie gevonden voor Cf₂, Cf₄, Cf₈, Cf₉ en Cf₁₁. Alle fysio's die op genotypen met Cf₄ konden groeien, groeiden ook op genotypen met Cf₈. Daarom kon geen ondersheid gemaakt worden tussen Cf₄ en Cf₈. De Poolse isolaten behoren tot de fysio's 4.11, 2.4.11 en 2.4.9.11. De gevolgen van het voorkomen van deze fysio's voor de tomateveredeling worden besproken.     

Allelic test proves genes Cf4 and Cf8 for resistance to Cladosporium fulvum (Fulvia fulva) on tomato to be undistinguishable

In an allelic test it was proven that the genes Cf₄ and Cf₈ for resistance toC. fulvum in tomato are undistinguishable, confirming a recent suggestion that Cf₈ does nott provide a novel source for resistance toC. fulvum.Samenvatting In een allelie-toets werd aangetoond dat de genen Cf₄ en Cf₈ voor resistentie tegenC. fulvum van tomaat niet te onderscheiden zijn. Hiermee werd een recente suggestie bevestigd, dat Cf₈ geen nieuwe resistentie tegenC. fulvum verschaft.     

番茄叶霉病高抗基因Cf-9的CAPS标记创建

根据番茄叶霉病高抗基因Cf-9的基因序列设计3对特异扩增引物,以近等基因系和本组的多重PCR检测材料为试材,扩增Cf-9基因1～2867 bp之间的单拷贝片段,3对引物分别扩增出560 bp,1 000 bp,1 080 bp的特异片段。分别用限制性内切酶TaqⅠ,HindⅢ,HinfⅠ,EcorⅠ酶切,限制性内切酶TaqⅠ酶切特异引物SCAR1扩增的560特异片段具有酶切多态性,抗病品种酶切出450 bp,330 bp,290 bp的特异片段,感病品种酶切出450 bp,290 bp的特异片段。初步建成番茄叶霉病高抗基因Cf-9的CAPS标记,经近等基因系及其杂交种检验,结果稳定可靠,可用于番茄Cf-9抗病基因辅助选育。     

Occurrence of a new race 2.9 of leaf mold of tomato in Japan

Leaf mold symptoms were found on tomato varieties carrying the Cf-9 resistance gene against Passalora fulva, the causal agent of leaf mold, in Japan in 2008. Disease symptoms and morphological characteristics of the isolates were similar to those of P. fulva. After inoculating a set of tomato differentials with the isolates, all isolates were identified as race 2.9 of P. fulva, previously unreported.     

The first occurrence of leaf mold of tomato caused by races 4.9 and 4.9.11 of <Emphasis Type="Italic">Passalora fulva</Emphasis> (syn. <Emphasis Type="Italic">Fulvia fulva</Emphasis>) in Japan

Tomato leaf mold caused by Passalora fulva was found on two tomato varieties carrying the Cf-9 gene in Japan, in 2007. The isolates obtained from Chiba and Fukushima were identified as race 4.9.11, and those from Gunma were races 4.9 or 4.9.11. This is the first report in Japan of tomato leaf mold caused by P. fulva strains that can overcome the Cf-9 gene.     

Diversity of Avr‐vnt1 and AvrSmira1 effector genes in Polish and Norwegian populations of Phytophthora infestans

The oomycete Phytophthora infestans, the cause of late blight, is one of the most important potato pathogens. During infection, it secretes effector proteins that manipulate host cell function, thus contributing to pathogenicity. This study examines sequence differentiation of two P. infestans effectors from 91 isolates collected in Poland and Norway and five reference isolates. A gene encoding the Avr‐vnt1 effector, recognized by the potato Rpi‐phu1 resistance gene product, is conserved. In contrast, the second effector, AvrSmira1 recognized by Rpi‐Smira1, is highly diverse. Both effectors contain positively selected amino acids. A majority of the polymorphisms and all selected sites are located in the effector C‐terminal region, which is responsible for their function inside host cells. Hence it is concluded that they are associated with a response to diversified target protein or recognition avoidance. Diversification of the AvrSmira1 effector sequences, which existed prior to the large‐scale cultivation of plants containing the Rpi‐Smira1 gene, may reduce the predicted durability of resistance provided by this gene. Although no isolates virulent to plants with the Rpi‐phu1 gene were found, the corresponding Avr‐vnt1 effector has undergone selection, providing evidence for an ongoing ‘arms race’ between the host and pathogen. Both genes remain valuable components for resistance gene pyramiding.     

A Large-Scale Survey of Races of Leptosphaeria maculans Occurring on Oilseed Rape in France

Nine avirulence genes (AvrLm1–AvrLm9) were identified in Leptosphaeria maculans, the causal agent of stem canker of oilseed rape (OSR), combinations of which could theoretically generate up to 512 different races of the fungus. L. maculans displays a high evolutionary potential to adapt to novel resistance genes as illustrated by the Rlm1 breakdown in France, where virulent populations became prevalent within three growing seasons. An improved knowledge of the race structure of the fungal population is therefore needed to ensure a better use of available major resistance genes. The objective of this study was to characterise the L. maculans population structure in France using a large-scale, rationalised sample of isolates. Experimental fields, planted with “trap plants” harbouring no major resistance gene, were sown at 20 locations. Single-pycnidium isolates were collected from leaf lesions that developed in early autumn and 1797 isolates were genotyped at Avr loci. The frequency of AvrLm6 and AvrLm7 was higher than 99%, whereas avrLm2 and avrLm9 alleles were fixed in the population. AvrLm1, AvrLm4, AvrLm5 and AvrLm8 were polymorphic. AvrLm3 isolates were detected at a very low frequency (less than 1%). Only 11 races were identified in France, with one race prevalent, namely Av5-6-7-(8) (i.e. virulent on Rlm1, Rlm2, Rlm3, Rlm4 and Rlm9), representing around 65% of the population. Disparities between the locations sampled were evident at all scales analysed. Some virulent races, such as those harbouring avrLm5, were present before the introduction of the corresponding resistance gene in the commercial OSR crop.     

Race 2 of Verticillium dahliae infecting tomato in Japan can be split into two races with differential pathogenicity on resistant rootstocks

Verticillium dahliae infecting tomato can be differentiated into races 1 and 2 based on differential pathogenicity on tomato cultivars carrying resistance gene Ve1. Although no commercial cultivars resistant to race 2 are available, race 2‐resistant rootstock cultivars Aibou and Ganbarune‐Karis have been bred in Japan. Nevertheless, the resistance of these rootstocks appears to be unstable in commercial tomato fields. Pathogenicity assays conducted under controlled conditions revealed that these rootstock cultivars are resistant to some isolates of race 2; this resistance is controlled by a single dominant locus, denoted by V2, based on segregation of resistance in F₂ populations from selfed rootstock cultivars. However, some other isolates of race 2 can overcome this resistance. Therefore it is proposed that the current race 2 of V. dahliae should be divided into two races, i.e. ‘race 2’ (nonpathogenic on Aibou) and ‘race 3’ (pathogenic on Aibou). The distribution of these races was surveyed in 70 commercial tomato fields in Hida, Gifu Prefecture, Japan. Race 3 was found in 45 fields, indicating that race 3 had already spread throughout the region. On the other hand, 25 fields had only race 2, and thus race 2‐resistant rootstocks would be effective for disease management in these fields. Races 2 and 3 could not be identified by genomic Southern hybridization probed with a telomere sequence, nor with previously reported race‐specific PCR assays. Elucidation of race‐determining mechanisms and development of methods for quick race identification should be made in future studies.     

球孢白僵菌定殖提高番茄对灰霉病抗性及其作用机理

为明确球孢白僵菌Beauveria bassiana定殖对番茄植株抗病性的影响及作用机理,采用灌根法将球孢白僵菌芽生孢子悬浮液接种于番茄植株内,并通过人工接种灰霉菌Botrytis cinerea评价球孢白僵菌定殖后番茄对灰霉病的抗性水平;检测灰霉菌胁迫下番茄植株不同位置叶片内球孢白僵菌的相对含量;测定番茄叶片内草酸氧化酶（oxalate oxidase,OXO）、几丁质酶（chitinase,CHI）和ATP合成酶（ATP synthase,atpA）3种抗病相关基因的表达量。结果表明,球孢白僵菌定殖能够提高番茄植株对灰霉病的抗性,接种灰霉菌第5天,番茄植株发病率、病斑直径和病情指数分别下降了61.6%、41.4%和26.4%;在灰霉菌胁迫下番茄植株内球孢白僵菌偏好于在病原菌感染位置定向聚集,并且引起植物抗病基因OXO、CHI和atpA的表达量上调。表明球孢白僵菌能通过内生定殖与植物互作提高植物抗病性,在植物病害生物防治领域有较大应用潜力。     

番茄与叶霉菌互作的分子机理

 番茄和叶霉菌(Cladosporium fulvum Cooke)系统是研究植物和病原物互作分子机理的模式系统。4个叶霉菌无毒基因已被克隆。这些无毒基因编码含偶数个半胱氨酸的小分子量蛋白,在叶霉菌毒性菌株中的存在与否及存在方式各不相同。Avr4具有几丁质结合活性;而Avr9可能在叶霉菌氮素代谢中起调节作用。7个具有功能的番茄抗叶霉菌Cf基因已被克隆。它们与其同源基因一起以基因簇形式存在于复合基因座中,其成员称为Hcr (homologues of C. fulvum resistance gene Cf)基因。Cf蛋白定位于细胞质膜,但主体在膜外,主要结构域为富含亮氨酸重复(LRR)和跨膜结构域。LRR重复单元数目以及N端序列决定了Cf蛋白对Avr的识别特异性。Cf对Avr的识别遵守"保卫"假说。在Cf-2对Avr2的识别系统中,蛋白酶Rcr3为"被保卫"蛋白。Cf识别Avr后迅速激活下游信号传导和防卫反应,包括过敏性反应的产生和氧化迸发,以及K⁺离子通道、各类蛋白激酶、SGT1、硫氧还蛋白及磷脂酸途径的活化。温度、湿度和光照等环境条件显著影响Cf介导的过敏性反应和抗病性。不同Cf对Avr的识别机理及其下游信号传导途径有显著差别。     

Local and systemic resistance to fungal pathogens triggered by an AVR9-mediated hypersensitive response in tomato and oilseed rape carrying the Cf-9 resistance gene

Tomato and transgenic oilseed rape plants expressing the Cf-9 resistance gene develop a hypersensitive response (HR) after injection of the corresponding Avr9 gene product. It was investigated whether induction of a HR conferred resistance to different fungal pathogens in tomato and oilseed rape. Induction of an AVR9 mediated HR at the pathogen infection site delayed the development of the biotrophs Oidium lycopersicum in tomato and Erysiphe polygoni in oilseed rape, but enhanced the development of the necrotrophs Botrytis cinerea and Alternaria solani in tomato and Sclerotinia sclerotiorum in oilseed rape. Interestingly, delayed fungal disease development was observed in plant tissues surrounding the HR lesion regardless of whether a necrotrophic or biotrophic pathogen was used. In tomato, AVR9 injection induced systemic expression of PR1, PR2 and PR3 defence genes but did not induce systemic resistance to O. lycopersicum, B. cinerea or A. solani. In oilseed rape, AVR9 injection temporarily induced systemic resistance to Leptosphaeria maculans and E. polygoni, but did not induce detectable systemic expression of PR1, PR2 or Cxc750. These results give new insights into the potential uses of an induced HR to engineer disease resistance.     

Analysis of Leptosphaeria maculans Race Structure in a Worldwide Collection of Isolates

ABSTRACT Leptosphaeria maculans, the causal agent of stem canker of oilseed rape, develops gene-for-gene interactions with its hosts. To date, eight L. maculans avirulence (Avr) genes, AvrLm1 to AvrLm8, have been genetically characterized. An additional Avr gene, AvrLm9, that interacts with the resistance gene Rlm9, was genetically characterized here following in vitro crosses of the pathogen. A worldwide collection of 63 isolates, including the International Blackleg of Crucifers Network collection, was genotyped at these nine Avr loci. In a first step, isolates were classified into pathogenicity groups (PGs) using two published differential sets. This analysis revealed geographical disparities as regards the proportion of each PG. Genotyping of isolates at all Avr loci confirmed the disparities between continents, in terms of Avr allele frequencies, particularly for AvrLm2, AvrLm3, AvrLm7, AvrLm8, and AvrLm9, or in terms of race structure, diversity, and complexity. Twenty-six distinct races were identified in the collection. A larger number of races (n = 18) was found in Australia than in Europe (n = 8). Mean number of virulence alleles per isolate was also higher in Australia (5.11 virulence alleles) than in Europe (4.33) and Canada (3.46). Due to the diversity of populations of L. maculans evidenced here at the race level, a new, open terminology is proposed for L. maculans race designation, indicating all Avr loci for which the isolate is avirulent.     

北京地区番茄灰霉病菌对嘧霉胺的抗药性检测

为了检测北京地区番茄灰霉病菌对嘧霉胺的抗药性,指导生产上科学用药,从北京12个区、县采集番茄灰霉病样150份,分离纯化获得109个番茄灰霉病菌(Botrytis cinerea)的单孢菌株,采用菌丝生长速率法测定了番茄灰霉病菌对嘧霉胺(pyrimethanil)的抗药性。所测菌株中嘧霉胺抗性菌株出现的比例高达82.57%,且以高抗菌株为主,占78.89%,抗性水平最高的菌株EC50是最敏感菌株的5 096倍;不同区县番茄灰霉病菌对嘧霉胺的抗药性存在差异,门头沟区、密云县、延庆县、怀柔区和平谷区、大兴、通州和顺义8个区县的高抗菌株所占比例达66.67%～100%,海淀、朝阳和房山3个区未检测到高抗菌株。上述结果说明北京地区番茄灰霉病菌对嘧霉胺的抗药性非常普遍,且以高抗菌株为主,生产上应替换新型杀菌剂防治番茄灰霉病。     

Variation of pathotypes and races and their correlations with clonal lineages in Verticillium dahliae

Understanding pathogenic variation in plant pathogen populations is key for the development and use of host resistance for managing verticillium wilt diseases. A highly virulent defoliating (D) pathotype in Verticillium dahliae has previously been shown to occur only in one clonal lineage (lineage 1A). By contrast, no clear association has yet been shown for race 1 with clonal lineages. Race 1 carries the effector gene Ave1 and is avirulent on hosts that carry resistance gene Ve1 or its homologues. The hypothesis tested was that race 1 arose once in a single clonal lineage, which might be expected if V. dahliae acquired Ave1 by horizontal gene transfer from plants, as hypothesized previously. In a diverse sample of 195 V. dahliae isolates from nine clonal lineages, all race 1 isolates were present only in lineage 2A. Conversely, all lineage 2A isolates displayed the race 1 phenotype. Moreover, 900‐bp nucleotide sequences from Ave1 were identical among 27 lineage 2A isolates and identical to sequences from other V. dahliae race 1 isolates in GenBank. The finding of race 1 in a single clonal lineage, with identical Ave1 sequences, is consistent with the hypothesis that race 1 arose once in V. dahliae. Molecular markers and virulence assays also confirmed the well‐established finding that the D pathotype is found only in lineage 1A. Pathogenicity assays indicated that cotton and olive isolates of the D pathotype (lineage 1A) were highly virulent on cotton and olive, but had low virulence on tomato.     

PCR-based differentiation of Fusarium oxysporum ff. sp. lycopersici and radicis-lycopersici and races of F. oxysporum f. sp. lycopersici

The pathogenic type (form and race) of Fusarium oxysporum, which generates wilt symptoms on tomato, was rapidly identified with a polymerase chain reaction (PCR)-based technique. We compared the partial nucleotide sequences of endo polygalacturonase (pg1) and exo polygalacturonase (pgx4) genes from isolates of F. oxysporum ff. sp. lycopersici (FOL) and radicis-lycopersici (FORL) from Japan and designed specific primer sets (uni, sp13, sp23, and sprl) based on the nucleotide differences that appeared among the pathogenic types. PCR with the uni primer set amplified a 670∼672-bp fragment from all isolates of FOL and FORL. With the sp13 primer set, an amplicon of 445 bp was obtained only from isolates of FOL race 1 and 3. With the sp23 primer set, a 518-bp fragment was obtained from isolates of FOL race 2 and 3. The sprl primer set yielded a 947-bp fragment from isolates of FORL, but not from FOL. A combination of amplifications with these primer sets effectively differentiated the pathogenic types of F. oxysporum in tomato.     

Biological and molecular characterization of tomato spotted wilt virus (TSWV) resistance-breaking isolates from Argentina

Tomato spotted wilt virus (TSWV) has been present in Argentina since 1938 and had limited sweet pepper and tomato production until the introduction of resistant cultivars bearing Tsw and Sw-5b genes. However, the wide use of TSWV-resistant pepper plants in La Plata Horticultural Belt (LPHB) triggered the emergence of resistance-breaking isolates (RB), increasing the economic impact of TSWV in pepper. This work characterized 11 natural RB pepper isolates from LPHB that have overcome the Tsw resistance gene in Capsicum sp. but are unable to break the Sw-5b-mediated resistance in tomato. Phylogenetic analysis of the N gene showed that the LPHB isolates are most closely related to isolates from Asia, indicating that Argentine TSWV isolates might have emerged from the Asian continent. The NSs sequence analysis reinforces the hypothesis that the appearance of an RB phenotype is a consequence of a number of different single amino acid substitutions spread along the NSs gene that lead to multiple independent evolutionary events. These results provide information on the current situation of the tospovirus–pepper/tomato pathosystems in LPHB, which represents a fundamental prerequisite to include these RB isolates in future screening programmes in order to select new and durable sources of resistance to TSWV in pepper.     

Characterization of elicitor activities of apoplastic fluids isolated from tomato lines infected with new races of Cladosporium fulvum

Apoplastic fluids were isolated from the near-isogenic lines Cf2, Cf4 and Cf5 of the tomato (Lycopersicon esculentum Mill.) cultivar ‘Moneymaker’ infected with a set of newCladosporium fulvum races 2.5, 2.4.11, 2.5.9, 2.4.5.11, 2.4.9.11 and 2.4.5.9.11 and a set of previously described races 2, 4, 5, 2.4., 2.4.5 and 2.4.5.9. Upon injection of the apoplastic fluids into leaves of the near-isogenic lines Cf0, Cf2, Cf4, Cf5 and Cf9 and the genotypes carrying Cf6 and Cf11 chlorosis or necrosis was observed in all plants that were resistant to the race that initially had been used to raise the apoplastic fluid, except in the genotype Cf11 when the races 4, 2.4, 2.4.5 and 2.4.5.9 from our own collection were involved. The latter races may contain virulence gene all as our collection had not yet been checked for presence of this virulence gene. Apoplastic fluids of all races induced chlorosis on the genotype carrying resistance gene Cf6, a gene which has not yet been overcome by any of the presently described races ofC. fulvum.Low pH non-denaturing polyacrylamide gel electrophoresis showed that apoplastic fluids of compatible interactions with the new races 2.5.9, 2.4.9.11 and 2.4.5.9.11 carrying the virulence gene a9, like the previously described race 2.4.5.9, did not contain the necrosis-inducing peptide, while the peptide was present in all races carrying avirulence gene A9. This indicates that this race-specific elicitor is strongly associated with avirulence gene A9 and is possibly its product as has been suggested previously.     

Characterization of phenotypic variants of Clavibacter michiganensis subsp. michiganensis isolated from Capsicum annuum

Phenotypic variants of Clavibacter michiganensis subsp. michiganensis (Cmm) were isolated from pepper fields and from pepper seeds during quarantine inspections. All strains isolated from pepper (pepper isolates) produced orange-coloured colonies with lower mucoidy than typical Cmm strains isolated from tomato (tomato isolates). However, the results of ELISA, fatty acid analysis, 16S rDNA sequencing, and PCR analysis showed that all pepper isolates were similar enough to be identified as Cmm. In addition to phenotypic variations, the pepper isolates showed different pathogenic and genetic characteristics from tomato isolates from the USA, Europe, or other countries. They could be clearly distinguished in terms of pathogenicity, as they showed increased pathogenicity to pepper but reduced pathogenicity to tomato. Tomato isolates caused strong wilting and canker in tomato, but caused only canker and no wilting in pepper and bell pepper. However, pepper isolates caused no wilting, even in tomato, and only caused canker in the three host plants. In addition, compared to tomato isolates, pepper isolates showed increased colonization efficiency and caused a greater reduction in shoot dry weight in pepper. Pepper and tomato isolates could be separated into two groups according to host origin on the basis of 16S rDNA and ITS sequence analysis. They also showed different rep-PCR genomic fingerprints. All pepper isolates showed higher cellulase activity than tomato isolates on M9CMC plates. However, two plasmid-borne virulence genes of Cmm, pat-1, and celA, were not detected in any pepper isolates by PCR. Furthermore, PCR for pathogenicity-related genes located on a pathogenicity island (PAI) revealed that all tomato isolates were positive for these genes, whereas the pepper isolates did not show any PCR products for the chpC, chpG, ppaA, or tomA genes. Therefore, we suggest that the pepper isolates may represent a separate Cmm population that has evolved within the limits of this host.     

六种三唑类杀菌剂对番茄叶霉病菌的毒力及其安全性和田间防效评价

为筛选出有效防治番茄叶霉病的药剂,采用生长速率法及平板涂布法测定6种三唑类杀菌剂对番茄叶霉病菌菌丝生长及分生孢子萌发和芽管伸长的毒力,评价其对番茄植株的安全性和对叶霉病的田间防效。结果表明,己唑醇、苯醚甲环唑、戊唑醇和氟硅唑对番茄叶霉病菌菌丝生长的毒力均较高,EC_(50)分别为0.50、0.55、0.80、2.42 mg/L,其次为腈菌唑和四氟醚唑,EC50分别为6.92、15.08 mg/L。6种杀菌剂抑制病菌分生孢子萌发及芽管伸长的作用均较弱,对芽管伸长的抑制活性高于对孢子萌发的抑制活性;戊唑醇和四氟醚唑抑制孢子萌发的作用相对较强,100 mg/L处理的抑制率为60%~70%,戊唑醇、四氟醚唑和己唑醇抑制芽管伸长的作用相对较强,100 mg/L处理的抑制率均在70%以上。己唑醇和戊唑醇200 mg/L处理番茄植株,显著抑制其株高,苯醚甲环唑和腈菌唑对其影响相对较小,这4种杀菌剂对番茄植株的叶色及形态均无明显影响;且这4种杀菌剂对番茄叶霉病的田间预防效果均高于治疗效果,其中150 mg/L己唑醇的预防效果和治疗效果均最高,分别为90.67%和85.58%;苯醚甲环唑的最低,300 mg/L时预防效果为80.16%,治疗效果为71.68%。