AvrE1 and HopR1 from Pseudomonas syringae pv. actinidiae are additively required for full virulence on kiwifruit

Pseudomonas syringae pv. actinidiae ICMP 18884 biovar 3 (Psa3) produces necrotic lesions during infection of its kiwifruit host. Bacterial growth in planta and lesion formation are dependent upon a functional type III secretion system (T3S), which translocates multiple effector proteins into host cells. Associated with the T3S locus is the conserved effector locus (CEL), which has been characterized and shown to be essential for the full virulence in other P. syringae pathovars. Two effectors at the CEL, hopM1 and avrE1, as well as an avrE1-related non-CEL effector, hopR1, have been shown to be redundant in the model pathogen P. syringae pv. tomato DC3000 (Pto), a close relative of Psa. However, it is not known whether CEL-related effectors are required for Psa pathogenicity. The Psa3 allele of hopM1, and its associated chaperone, shcM, have diverged significantly from their orthologs in Pto. Furthermore, the CEL effector hopAA1-1, as well as a related non-CEL effector, hopAA1-2, have both been pseudogenized. We have shown that HopM1 does not contribute to Psa3 virulence due to a truncation in shcM, a truncation conserved in the Psa lineage, probably due to the need to evade HopM1-triggered immunity in kiwifruit. We characterized the virulence contribution of CEL and related effectors in Psa3 and found that only avrE1 and hopR1, additively, are required for in planta growth and lesion production. This is unlike the redundancy described for these effectors in Pto and indicates that these two Psa3 genes are key determinants essential for kiwifruit bacterial canker disease.     

猕猴桃溃疡病抗性育种研究进展

猕猴桃细菌性溃疡病是一种危害世界猕猴桃生产的毁灭性病害,目前尚未有有效的防治办法。培育抗性品种是保证猕猴桃产业健康发展的重要途径之一,猕猴桃溃疡病抗性育种成为近年来猕猴桃研究的热点。但是,目前大部分猕猴桃种质资源对溃疡病的抗性不明,限制了猕猴桃优异抗性种质资源的发掘和利用。虽然人们发展出了一些猕猴桃溃疡病抗性鉴定和评价方法,但是使用效果并不理想,存在较大的局限性,鉴定的准确性和稳定性还有待提高。该文针对猕猴桃溃疡病抗性育种中的几个方面,如抗性材料的选育(现有品种的抗性、抗性砧木研究和野生抗溃资源等),抗性鉴定和评价技术(大田鉴定、活体或离体鉴定等)及抗性机理研究等进行综述,并针对存在的问题,提出建设性意见。在猕猴桃溃疡病抗性育种过程中,最关键的是要建立一个科学、系统的溃疡病抗性评价体系,以对猕猴桃种质资源进行大规模的抗性普查和评估,在此基础上充分利用种间杂交和工程育种技术加快抗性育种进程,并以此带动猕猴桃溃疡病抗性机理的深入研究和抗病基因的挖掘和利用等,旨在从根本上解决猕猴桃生产中受溃疡病困扰这一关键难题,促进猕猴桃产业绿色、健康和可持续性发展。     

In vitro breeding – shortcut to Pseudomonas syringae pv. actinidiae (Psa) tolerant kiwifruit

Pseudomonas syringae pv. actinidiae (Psa) causes the bacterial canker disease on kiwifruit vines. The disease outbreak has been reported in several countries worldwide, including New Zealand. Here, we briefly reviewed the current situation of Psa infection of kiwifruit vines in New Zealand, the effects of Psa on the New Zealand’s kiwifruit industry, and the disease control and breeding programmes undertaken in response to the outbreak of Psa in New Zealand. Then the methodology of an alternative breeding approach or in vitro breeding, which is a non-GM approach to obtain useful plant tissue culture-derived genetic variation in crop plants, was discussed. As a specific example of potential application of in vitro breeding, a novel plant breeding project idea based on the elemental defence mechanism is to generate Cu/Zn tolerant kiwifruit varieties that exhibit improved Psa tolerance.     

Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae biovar 3 to Actinidia arguta

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is not a host of Psa3. Resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 51 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors–AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a –suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars.     

噬菌体鸡尾酒对猕猴桃溃疡病的田间防治效果及叶际细菌群落结构的影响

【背景】噬菌体鸡尾酒可作为一种杀灭猕猴桃溃疡病病原菌(Pseudomonassyringaepv.actinidiae, Psa)的生物制剂,但关于噬菌体鸡尾酒在田间的防治效果和对猕猴桃植株叶际内生细菌群落结构影响的研究依然较少。【目的】探究噬菌体鸡尾酒在田间防控猕猴桃溃疡病的效果,以及噬菌体鸡尾酒对猕猴桃茎内叶际细菌微生态的影响。【方法】使用猕猴桃溃疡病病原菌感染健康植株,对比施用噬菌体鸡尾酒和传统铜制剂后溃疡病的发病情况,利用高通量测序技术分析猕猴桃叶际内生细菌群落结构的变化。【结果】与铜制剂相比,噬菌体鸡尾酒可更有效地控制猕猴桃溃疡病,改变叶际细菌群落的丰富度与多样性,增强群落结构的稳定性,改善群落物种功能基因丰度情况,一定程度使叶际细菌群落恢复至健康状态。【结论】噬菌体鸡尾酒在杀灭病原菌的同时具有良好的微生态调节功能,在猕猴桃溃疡病的生物防治中具有巨大的应用潜力。     

In vitro antimicrobial activity of plant extracts against Pseudomonas syringae pv. actinidiae causal agent of bacterial canker in kiwifruit

Abstract

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker of kiwifruit, is considered the main pathogen of yellow-, green- and red-fleshed kiwifruit. All major kiwifruit producing countries in the world have been affected by this bacterial pathogen, leading to substantial economic losses. The control of bacterial canker of kiwifruit is based only on preventive methods or on the use of copper compounds that can cause phytotoxicity problems. In this study, the in vitro antibacterial activity of seven different plant extracts against eight Psa strains has been evaluated. The inhibition of 100% of the Psa growth was observed, after 24?h, for the extracts of Polygonum cuspidatum roots (POL-roots), Hypericum perforatum roots elicited with chitosan oligosaccharides (HYP-COS roots) and non-fermented grape pomace (ITA-pomace). The strongest antibacterial activity was exhibited by POL-roots, with a geometric mean of minimum inhibitory concentration of 100% of growth (GMMIC₁₀₀) of 105.11 µg/mL after 24?h, and with a GMMIC₁₀₀ value of 148.65 µg/mL after 48?h. Moreover, POL-roots extract showed the best bactericidal activity with a GMMBC of 210.22 µg/mL. No phytotoxic activity was observed up to 15 days in the leaves of Actinidia chinensis “Belen” treated with plant extracts at 500 µg/mL.     

Pseudomonas syringae pv. actinidiae detection in kiwifruit plant tissue and bleeding sap

The rapid spreading of the disease during last few years highlighted the need of a quick, sensitive and reliable method for Pseudomonas syringae pv. actinidiae (Psa) detection, to find possible inoculum sources and limit the pathogen spreading. A PCR method, using new primers designed on the gene encoding a putative outer membrane protein P1, was developed to detect Psa in symptomatic and asymptomatic tissue; a nested‐PCR was also applied. Bleeding sap samples, collected in early spring from orchards with symptomatic and asymptomatic trees, were used both for PCR assays and for pathogen isolation and identification. The PCR and nested PCR methods were able to detect Psa presence at very low concentration from plant and pollen extracts; RFLP analyses with BclI on PCR and nested PCR amplicons confirmed the assay specificity, while the digestion with BfmI and AluI allowed to discriminate Psa strains isolated before 2008 from those isolated after 2008. Furthermore, the PCR and nested PCR on crude bleeding sap samples detected the presence of the pathogen in 3 and 5 of the 15 assayed samples, respectively. Direct isolation from the same samples and bacterial identification confirmed the results of molecular analysis.     

Impact of selection and breeding on the genetic diversity in Douglas-fir

Genetic changes following domestication of Douglas-fir were studied using isozyme data derived from two generations of seed orchards and their 49 wild progenitor populations. In addition, the breeding, production, and infusion populations used in the seed orchards were compared to their wild counterparts. Several parameters of gene diversity were measured (number of alleles per locus N _a, per cent of polymorphic loci PLP, and expected heterozygosity H, and population divergence D). These measures were similar or higher in the domesticated populations compared to their natural progenitors, indicating that early selection and breeding of a highly polymorphic species does not significantly reduce genetic variation. The two generations of seed orchards also did not differ, indicating that genetic variation may remain stable over future generations of forest plantations. Interestingly, compared to the natural populations, heterozygosity was higher in the seed orchards, probably due to pooling of widely distributed natural populations; however, rare localized or private alleles seemed to be less frequent in the domesticated populations. Differentiation values were not significant between the first generation orchards and the natural populations, but significant differences were observed between the second generation orchards and the wild progenitor populations, probably due to the interbreeding that forms the advanced generation seed orchards.     

Genetic Diversity in Monilinia laxa Populations in Peach Orchards in Spain

One‐hundred and forty‐four random amplified polymorphic DNA markers, of which 59 were polymorphic and 85 monomorphic, were used to assess the genetic diversity and to study the structure of Monilinia laxa populations in Spain. Twenty‐one isolates collected from several orchards (subpopulations), in various years and in various hosts, were used. The analysis of population structure revealed that genetic diversity within orchards (H_S) accounted for 97% of the total genetic diversity (H_T), while genetic diversity among the orchards represented only 3%. The relative magnitude of gene differentiation between subpopulations (G_ST) and the estimate of the number of migrants per generation (N_m) averaged 0.032 and 15.1 respectively. The results obtained in dendrograms were in accordance with the gene diversity analysis. Grouping of isolates in the dendrogram was independent of whether they came from the same or different orchards. There was no relationship between clustering among isolates from distinct years and hosts. The relative importance of several evolutionary forces in populations of M. laxa is discussed, together with implications for the management of brown rot.     

A framework to gauge the epidemic potential of plant pathogens in environmental reservoirs: the example of kiwifruit canker

New economically important diseases on crops and forest trees emerge recurrently. An understanding of where new pathogenic lines come from and how they evolve is fundamental for the deployment of accurate surveillance methods. We used kiwifruit bacterial canker as a model to assess the importance of potential reservoirs of new pathogenic lineages. The current kiwifruit canker epidemic is at least the fourth outbreak of the disease on kiwifruit caused by Pseudomonas syringae in the mere 50 years in which this crop has been cultivated worldwide, with each outbreak being caused by different genetic lines of the bacterium. Here, we ask whether strains in natural (non‐agricultural) environments could cause future epidemics of canker on kiwifruit. To answer this question, we evaluated the pathogenicity, endophytic colonization capacity and competitiveness on kiwifruit of P. syringae strains genetically similar to epidemic strains and originally isolated from aquatic and subalpine habitats. All environmental strains possessing an operon involved in the degradation of aromatic compounds via the catechol pathway grew endophytically and caused symptoms in kiwifruit vascular tissue. Environmental and epidemic strains showed a wide host range, revealing their potential as future pathogens of a variety of hosts. Environmental strains co‐existed endophytically with CFBP 7286, an epidemic strain, and shared about 20 virulence genes, but were missing six virulence genes found in all epidemic strains. By identifying the specific gene content in genetic backgrounds similar to known epidemic strains, we developed criteria to assess the epidemic potential and to survey for such strains as a means of forecasting and managing disease emergence.     

Genetic specificity and potential for local adaptation between dengue viruses and mosquito vectors

Background  

Several observations support the hypothesis that vector-driven selection plays an important role in shaping dengue virus (DENV) genetic diversity. Clustering of DENV genetic diversity at a particular location may reflect underlying genetic structure of vector populations, which combined with specific vector genotype × virus genotype (G × G) interactions may promote adaptation of viral lineages to local mosquito vector genotypes. Although spatial structure of vector polymorphism at neutral genetic loci is well-documented, existence of G × G interactions between mosquito and virus genotypes has not been formally demonstrated in natural populations. Here we measure G × G interactions in a system representative of a natural situation in Thailand by challenging three isofemale families from field-derived Aedes aegypti with three contemporaneous low-passage isolates of DENV-1.     

Amino acid substitution in P3 of Soybean mosaic virus to convert avirulence to virulence on Rsv4‐genotype soybean is influenced by the genetic composition of P3

The modification of avirulence factors of plant viruses by one or more amino acid substitutions converts avirulence to virulence on hosts containing resistance genes. Limited experimental studies have been conducted on avirulence/virulence factors of plant viruses, in particular those of potyviruses, to determine whether avirulence/virulence sites are conserved among strains. In this study, the Soybean mosaic virus (SMV)–Rsv4 pathosystem was exploited to determine whether: (i) avirulence/virulence determinants of SMV reside exclusively on P3 regardless of virus strain; and (ii) the sites residing on P3 and crucial for avirulence/virulence of isolates belonging to strain G2 are also involved in virulence of avirulent isolates belonging to strain G7. The results confirm that avirulence/virulence determinants of SMV on Rsv4‐genotype soybean reside exclusively on P3. Furthermore, the data show that sites involved in the virulence of SMV on Rsv4‐genotype soybean vary among strains, with the genetic composition of P3 playing a crucial role.     

Genetic Heterogeneity in a Natural Population of Acanthamoeba polyphaga from Soil,an Isoenzyme Analysis1

Acanthamoeba polyphaga, a free-living, bacterial feeder found in freshwater and soil, reproduces asexually and is morphologicaly distinguishable from other acanthamoebae. Isoenzyme analyses were done on 15 random, clonal isolates from soil. Electrophoretic patterns indicated that enzyme bands occurred in clusters consistent with that of a diploid organism. The data indicates that natural populations of A. polyphaga have a greater genetic diversity than laboratory isolates of other amoebae, resembling the heterogeneity observed for natural populations of bacteria.     

Evaluation of an in vitro cell assay to select attenuated bacterial mutants of Aeromonas hydrophila and Edwardsiella tarda to channel catfish

Aims: To evaluate the feasibility of using an in vitro cell assay to select attenuated bacterial mutants. Methods and Results: Using catfish gill cells G1B, the feasibility of using an in vitro assay instead of in vivo virulence assay using live fish to select attenuated bacterial mutants was evaluated in this study. Pearson correlation analysis between in vitro virulence to G1B cells and in vivo virulence of Aeromonas hydrophila and Edwardsiella tarda revealed that there was a significant correlation between the two (r = ?0·768, P value = 3·7 × 10^?16). Conclusions: The in vitro cell assay might be initially used to screen large quantities of bacteria to select attenuated mutants of catfish pathogens. Significance and Impact of the Study: The in vitro cell assay using catfish gill cells to identify attenuated mutants of catfish pathogens will reduce cost involved in the in vivo virulence assay that requires many fish and aquariums.     

Plasmid composition and the chpG gene determine the virulence level of Clavibacter capsici natural isolates in pepper

The gram-positive bacterial species Clavibacter capsici causes necrosis and canker in pepper plants. Genomic and functional analyses of C. capsici type strain PF008 have shown that multiple virulence genes exist in its two plasmids. We aimed to identify the key determinants that control the virulence of C. capsici. Pepper leaves inoculated with 54 natural isolates exhibited significant variation in the necrosis. Six isolates showed very low virulence, but their population titres in plants were not significantly different from those of the highly virulent isolates. All six isolates lacked the pCM1_Cc plasmid that carries chpG, which has been shown to be required for virulence and encodes a putative serine protease, but two of them, isolates 1,106 and 1,207, had the intact chpG elsewhere in the genome. Genomic analysis of these two isolates revealed that chpG was located in the pCM2_Cc plasmid, and two highly homologous regions were present next to the chpG locus. The chpG expression in isolate 1,106 was not induced in plants. Introduction of chpG of the PF008 strain into the six low-virulence isolates restored their virulence to that of PF008. Our findings indicate that there are at least three different variant groups of C. capsici and that the plasmid composition and the chpG gene are critical for determining the virulence level. Moreover, our findings also indicate that the virulence level of C. capsici does not directly correlate with bacterial titres in plants.