茄青枯假单胞菌一个毒性基因突变体的表型分析

花生植株致病性试验表明分离自花生的茄青枯假单胞菌菌株T2015的ORF3突变体T2135/R在花生上的致病性显著降低,说明该基因与病菌在花生上致病有关。T2135/R在培养基中生长繁殖与野生型T2015没有显著差别,T2135/R产生的胞外植物细胞壁降解酶类如纤维素酶、淀粉酶和蛋白酶与T2015相比没有显著区别,说明该基因与病菌在培养基中的生长繁殖、病菌的胞外植物细胞壁降解酶的产生无关。     

Pathogenic characterisation of strains of Ralstonia solanacearum from Ethiopia and influence of plant age on susceptibility of hosts against R. solanacearum

Journal of Plant Diseases and Protection - Ralstonia solanacearum is a diverse species that differs in host range and reaction on tobacco leaf and can be differentiated into races based on its host...     

Alteration of gene expression profile in the roots of wild diploid Arachis duranensis inoculated with Ralstonia solanacearum

Bacterial wilt caused by Ralstonia solanacearum is a serious disease of peanut (Arachis hypogaea) in China. However, the molecular basis of peanut resistance to R. solanacearum is poorly understood. Arachis duranensis, a wild diploid species of the genus Arachis, has been proven to be resistant to bacterial wilt, and thus holds valuable potential for understanding the mechanism of resistance to bacterial wilt and genetic improvement of peanut disease resistance. Here, suppression subtractive hybridization (SSH) and macroarray hybridization were employed to detect differentially expressed genes (DEGs) in the roots of A. duranensis after R. solanacearum inoculation. A total of 317 unique genes were obtained, 265 of which had homologues and functional annotations. KEGG analysis revealed that a large proportion of these unigenes are mainly involved in the biosynthesis of phytoalexins, particularly in the biosynthetic pathways of terpenoids and flavonoids. Subsequent real‐time polymerase chain reaction (PCR) analysis showed that the terpenoid and flavonoid synthesis‐related genes showed higher expression levels in a resistant genotype of A. duranensis than in a susceptible genotype, indicating that the terpenoids and flavonoids probably played a fundamental role in the resistance of A. duranensis to R. solanacearum. This study provides an overview of the gene expression profile in the roots of wild Arachis species in response to R. solanacearum infection. Moreover, the related candidate genes are also valuable for the further study of the molecular mechanisms of resistance to R. solanacearum.     

Effect of calcium nutrition on resistance of tomato against bacterial wilt induced by Ralstonia solanacearum

This study investigated the effect of calcium nutrition on tomato bacterial wilt caused by Ralstonia solanacearum and the regulation of resistance mechanisms. Plants cultured in nutrient solution with calcium concentrations of 0.5, 5.0, and 25.0 mM, were inoculated with R. solanacearum by the root dip method. Severity of disease development, Ca concentration in tomato root and shoot tissues, hydrogen peroxide (H₂O₂) concentration, peroxidase (POD, EC 1.11.1.7) and polyphenol oxidase (PPO, EC 1.10.3.2) in tomato leaves were analyzed. Disease severities of low, medium and high Ca treatments were 100 %, 77.1 % and 56.8 % respectively. Plant growth in high Ca treatment was significantly better than those in low Ca treatment in height, stem diameter and biomass. Tomato plants absorbed significantly more Ca in roots and shoots as the level of Ca in the nutrient solution increased. In addition, H₂O₂ level in high Ca treatment rose faster and reached a higher peak with 10.86 μM gFW^?1(31.32 % greater than medium Ca plants). The activities of POD and PPO also have a greater increase in high Ca treatment with 99.09 U gFW^?1 and 107.24 U gFW^?1 compared to 40.70 U gFW^?1 and 77.45 U gFW^?1 in low Ca treatment. A negative correlation was found between Ca concentration, level of H₂O₂, POD, PPO in tomato, and disease severity, indicating that they played an important role in resistance of tomato to this disease. These results suggested that Ca was involved in the regulation of H₂O₂ concentration, and activity of POD and PPO in tomato.     

番茄枯萎病和青枯病拮抗细菌的筛选、评价与鉴定

从宁夏银川、江苏沭阳和福建厦门的番茄、辣椒、西瓜等作物根际土壤中,分离纯化获得367株细菌菌株。以番茄枯萎病菌Fusarium oxysporum f.sp.lycopersici和番茄青枯病菌Ralstonia solanacearum为靶标菌,从367株菌株中筛选出对两种病菌皆具有很强拮抗作用的菌株22株。拮抗细菌抑菌物质的研究结果表明：22株拮抗细菌均能分泌蛋白酶;不能分泌几丁质酶;3株细菌能分泌纤维素酶;3株细菌能分泌嗜铁素。盆栽试验结果表明：拮抗细菌PTS-394对番茄枯萎病和青枯病的防效最高,分别为77.4%和80%;菌株H-70、L-1和SJ-280对番茄枯萎病和青枯病的防效均大于60%。对上述4株拮抗细菌进行16S rRNA种属鉴定,均为枯草芽孢杆菌Bacillus subtilis。     

Validation and use of a qPCR protocol to quantify the spread of Ralstonia solanacearum in susceptible and resistant eucalypt plants

Bacterial wilt caused by Ralstonia solanacearum is a serious disease of eucalypt in humid and high temperature areas worldwide. Spreading of the bacterium in the field or to other nurseries occurs mainly by symptomless infected plant material. The use of pathogen-free propagating material as well as planting of resistant genotypes are currently the only strategies used for disease control. Therefore, a reliable and sensitive method for detection of low titres of R. solanacearum in infected plant tissue is essential for the success of management programmes. In this work, we adapted an efficient intercalating dye-based real-time PCR protocol to detect the bacterium in symptomless eucalypt plants as well as to investigate its movement in eucalypt clones CLR172 and CLR371, which exhibit resistant and susceptible phenotypes, respectively. We found that the bacterium translocates acropetally and basipetally in inoculated but symptomless cuttings of the resistant clone, as in cuttings of the susceptible clone displaying symptoms. Nevertheless, a smaller concentration of bacterial DNA was detected in tissues of the resistant clone. Mature biofilms occluding the xylem vessels were present in the susceptible clone whereas only single cells or small aggregates were observed in the resistant clone. This work contributes to improve our knowledge of the colonization process of R. solanacearum in eucalypt clones with different levels of susceptibility and to understand how the defence mechanisms against bacterial wilt in Eucalyptus work. Our findings could aid in the selection of the most resistant eucalypt clones to be used in wilt disease management programmes.     

Involvement of a vascular hypersensitive response in quantitative resistance to Ralstonia solanacearum on tomato rootstock cultivar LS‐89

Ralstonia solanacearum causes bacterial wilt disease in Solanaceae spp. Expression of the Phytophthora inhibitor protease 1 (PIP1) gene, which encodes a papain‐like extracellular cysteine protease, is induced in R. solanacearum‐inoculated stem tissues of quantitatively resistant tomato cultivar LS‐89, but not in susceptible cultivar Ponderosa. Phytophthora inhibitor protease 1 is closely related to Rcr3, which is required for the Cf‐2‐mediated hypersensitive response (HR) to the leaf mould fungus Cladosporium fulvum and manifestation of HR cell death. However, up‐regulation of PIP1 in R. solanacearum‐inoculated LS‐89 stems was not accompanied by visible HR cell death. Nevertheless, upon electron microscopic examination of inoculated stem tissues of resistant cultivar LS‐89, several aggregated materials associated with HR cell death were observed in xylem parenchyma and pith cells surrounding xylem vessels. In addition, the accumulation of electron‐dense substances was observed within the xylem vessel lumen of inoculated stems. Moreover, when the leaves of LS‐89 or Ponderosa were infiltrated with 10⁶ cells mL^?1 R. solanacearum, cell death appeared in LS‐89 at 18 and 24 h after infiltration. The proliferation of bacteria in the infiltrated leaf tissues of LS‐89 was suppressed to approximately 10–30% of that in Ponderosa, and expression of the defence‐related gene PR‐2 and HR marker gene hsr203J was induced in the infiltrated tissues. These results indicated that the response of LS‐89 is a true HR, and induction of vascular HR in xylem parenchyma and pith cells surrounding xylem vessels seems to be associated with quantitative resistance of LS‐89 to R. solanacearum.     

Effect of a Soil Amendment on the Survival of Ralstonia solanacearum in Different Soils

ABSTRACT The effect of a soil amendment (SA) composed of urea (200 kg of N per ha) and CaO (5,000 kg/ha) on the survival of Ralstonia solanacearum in four Philippine soils was investigated in a series of laboratory experiments. Within 3 weeks after application, the SA either caused an initial decrease, a final decline, or no change in the pathogen population, depending on the particular soil type. An initial decrease occurred in a soil with a basic pH and resulted in a significantly (P < 0.001) lower pathogen population immediately and at 1 week after amending the soil. This decrease was probably due to the high pH in the soil during urea hydrolysis. A final decline in the R. solanacearum population after 3 weeks occurred in two soils in which nitrite accumulated after 1 week. In these soils, no decline in bacterial levels occurred when nitrite formation was inhibited by 2-chloro-6-trichloromethylpyridine. In the soil with low pH, no nitrite accumulated and the R. solanacearum population did not decline. The suppressive effects of pH and nitrite on R. solanacearum growth were confirmed by in vitro experiments. Ammonium reduced the growth of R. solanacearum, but was not suppressive. Interactions of pH with ammonium and nitrite also occurred, whereby ammonium reduced growth of R. solanacearum only at pH 9 and nitrite was suppressive only at pH 5. Nitrate had no effect on R. solanacearum growth in vitro.     

拮抗青枯劳尔氏菌的荧光假单胞菌SN15-2分离鉴定及其生防能力分析

为了筛选出对番茄青枯病具有较好防效的生防菌,采用皿内测定法从上海地区的番茄青枯病自然衰退土壤中,分离到一株对番茄青枯病有很强抑制作用的菌株SN15-2,并进行了分子鉴定和对荧光假单胞菌SN15-2产抗生素能力和定殖能力测定。结果表明,菌株SN15-2为荧光假单胞菌(Pseudomonas fluorescens)。其菌株能产生2,4-二乙酰基间苯三酚(2,4-DAPG)、硝吡咯菌素(PRN)、藤黄绿脓菌素(PLT)。同时,可以产生HCN、噬铁素,能够形成生物膜。SN15-2在施入番茄根际后的前20 d定殖数量减少,20 d后基本稳定,60 d时,在干土中的定殖数量可达3.67×105cfu/g。盆栽防效分析表明,荧光假单胞菌SN15-2对番茄青枯病防效达到46.58%。     

紫油厚朴叶提取物对马铃薯青枯病菌的抑制作用及防病效果

采用快速萃取法提取了紫油厚朴叶中的有效成分,用其提取物对马铃薯青枯病菌进行了抑菌作用及马铃薯青枯病的盆栽防治试验研究。结果表明:紫油厚朴叶提取物在浓度为含厚朴总酚0.6、0.3、0.2、0.1 mg/mL时对马铃薯青枯病菌均表现较强的抑菌活性;厚朴叶提取物对受试的马铃薯青枯病菌的最低抑菌浓度(MIC)为厚朴总酚0.1 mg/mL;盆栽防治试验效果亦很好。     

Identification and use of a wild plant with antimicrobial activity against Ralstonia solanacearum, the cause of bacterial wilt of potato

Fresh aerial tissue and roots of 14 wild plants in Okinawa prefecture were investigated for their antimicrobial activity against Ralstonia solanacearum , which causes bacterial wilt of potato. A 70% aqueous ethanol extract of fresh aerial tissue of Geranium carolinianum L. showed strong antimicrobial activity against R. solanacearum . This extract also showed antimicrobial activity against the pathogens causing common scab of potato and soil rot of sweet potato. The antimicrobial substance could be extracted with hot water, and was effective against R. solanacearum in soil. In the field test, a treatment combining incorporation of dried aerial tissue into the soil and solarization was highly effective for control of bacterial wilt of potato. These findings suggest that G. carolinianum L. could be used as a biological agent for the control of bacterial wilt of potato.     

番茄青枯病病原菌拮抗菌株的筛选及其田间防控作用研究

从番茄青枯病发病严重田块的健康植株根际土壤中分离筛选得到2株高效拮抗菌株,命名为W12和W118,经16SrDNA基因鉴定均属芽胞杆菌属;用PCR扩增的方法扩增脂肽类抗生素合成基因,结果表明W12和W118含有合成bacillomycin、iturin和fengycin三种抗生素的基因;将2株拮抗菌用于田间试验,结果表明混合菌株防控效果最好,3次灌菌后防控效果达到62.3%,单独施用菌株W118较单独施用W12防控效果好,3次灌菌后防控效果达到56.7%。     

Selection of virulent Meloidogyne individuals within mixed isolates by continuous cultivation on a Mi gene resistant tomato genotype

Journal of Plant Diseases and Protection - Two isolates of Meloidogyne spp. from two districts (Thrace and Crete) of Greece have been identified as M. arenaria and M. incognita using perineal...     

Ralstonia solanacearum as a potato pathogen in Serbia: Characterization of strains and influence on peroxidase activity in tubers

Since 2011, the outbreaks of brown rot caused by Ralstonia solanacearum race 3, biovar 2, phylotype IIB-1 (R3/B2/PIIB-1) have significantly compromised potato production in Serbia. During 6 years of monitoring (2013–2018) among 3,524 potato tuber samples, 344 were found positive for brown rot disease. R. solanacearum R3/B2/PIIB-1 was isolated from seven cultivars among 12 monitored, and in five localities among 17 monitored. Cultivar Lady Claire was found to have the highest disease frequency (31.98%). A total of 78 isolates were identified by R. solanacearum-specific primer pairs (PS-1/PS-2 and OLI-1/Y-2), as well as the following tests: restriction fragment length polymorphism analysis, biovar determination, immunofluorescence, biochemical analysis, and pathogenicity. The genetic composition of 36 selected isolates assessed using multilocus sequence analysis with seven genes (adk, gapA, gdhA, gyrB, ppsA, hrpB, and fliC) showed that all isolates originating from Serbian potato were homogeneous. By using the TCS algorithm of concatenated sequences to get insight into the phylogeography of isolates and other R. solanacearum strains deposited in the NCBI database, we showed that their origin is undetermined. Peroxidase (POD) activity was measured in brown rotted potato tubers. A positive correlation was found between POD activity and disease severity rated on the analysed tubers. In general, POD activity increased by 2–22 times in vascular necrotic tissues compared to non-necrotic ones, and depended on disease severity but not on cultivar. Native polyacrylamide gel electrophoresis analysis of POD profiles resulted in a total of 10 distinct POD isoforms, of which PODs 3–5 were highly intensified in response to R. solanacearum.     

Phylogenetic relationships of Ralstonia solanacearum species complex strains from Asia and other continents based on 16S rDNA, endoglucanase, and hrpB gene sequences

The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AY464950 to AY465050     

Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum (Smith) Yabuuchi et al. race 3 are the causal agents of ring-rot and brown-rot of potato respectively. These diseases represent a serious threat to potato production in temperate climates. Both bacteria are listed as A2 pests in the EPPO region and as zero-tolerance quarantine organisms in the European Union. All the detection tests developed so far were only focused on the detection of a single pathogen while the absence of both bacteria has to be certified in the seed tubers. We have therefore developed a new multiplex real-time PCR assay to simultaneously detect both bacteria in a single assay. Additionally, the reliability of this molecular diagnostic test has been improved by the simultaneous amplification of an internal control, corresponding to a potato gene co-extracted from the sample. The polyvalence and the specificity of each set of bacterial primers and probes were evaluated on more than 90 bacterial strains. The limit of detection of this triplex real-time protocol was similar to those observed with other molecular protocols previously developed for the individual detection of one of these bacteria. A concordance of 100 % was obtained in a blind test mimicking the routine application of the technology. In conclusion, this new protocol represents a straightforward and convenient method potentially adapted to primary screening of potato tubers.     

Validation of four real‐time TaqMan PCRs for the detection of Ralstonia solanacearum and/or Ralstonia pseudosolanacearum and/or Clavibacter michiganensis subsp. sepedonicus in potato tubers using a statistical regression approach

A new DNA extraction method and a new multiplex real‐time TaqMan PCR test for detection of Ralstonia solanacearum, Ralstonia pseudosolanacearum and Clavibacter michiganensis subsp. sepedonicus in asymptomatic potato tubers are presented. This new multiplex PCR and three published TaqMan PCRs for detection of R. solanacearum and/or R. pseudosolanacearum and/or R. syzygii spp. and/or C. michiganensis subsp. sepedonicus were validated using linear regression analysis for estimating the Ct values and its variation at 5 × 10³ bacteria mL^?1. The three published PCRs that have been validated are Massart et al. (2014, detecting R. solanacearum and C. michiganensis subsp. sepedonicus), Weller et al. (1999, detecting R. solanacearum, R. pseudosolanacearum and R. syzygii spp.) and Gudmestad et al. (2009, detecting C. michiganensis subsp. sepedonicus). All tested PCRs were fit for purpose for their target organisms. The PCR tests have different target genes, allowing one of the sets to be used as first screening test and another as second screening test for the detection of R. solanacearum and/or R. pseudosolanacearum and/or C. michiganensis subsp. sepedonicus in asymptomatic potato tubers.     

Development of Myzus persicae on a partially resistant and on a susceptible genotype of lettuce (Lactuca sativa) in relation to plant age

The relationship between lettuce (Lactuca sativa) andMyzus persicae is influenced by internal and external factors. For the improvement of screening methods and the evaluation of the resistances found, a better knowledge of these factors is wanted.In five experiments the influence of plant age on resistance level was investigated for a partially resistant and a susceptible cultivar. Criteria for resistance were: remaining percentage of aphids (RPA), aphid developmental rate, insect biomass, and larvae production.It appeared that the aphids developed faster and grew better on older plants compared with younger plants, resulting in a decrease of overall level of resistance. The absolute differences between the susceptible and the resistant genotype for parameters such as biomass increased if plants were older and aphids were allowed to utilize all parts of the plant. It is concluded that with older plants (plant age e.g. 30–40 days) a better discriminative selection can be carried out.Samenvatting De relatie tussen sla enMyzus persicae wordt door zowel interne als externe factoren beïnvloed. Voor de verbetering van toetsmethoden en voor de evaluatie van gevonden resistenties, is meer kennis omtrent deze factoren noodzakelijk.In vijf proeven (I–V) werd de invloed van leeftijd van de plant op het resistentieniveau onderzocht bij een partiëel resistent en een vatbaar ras. Criteria voor resistentie waren: overblijvend percentage luizen 7 dagen na inoculatie, ontwikkelingssnelheid van de luizen, de insekt-biomassa en de larvenproduktie.Het bleek dat de groei en de ontwikkeling van de luizen op oudere planten beter was dan op jongere. Dit resulteerde in een afname van het resistentieniveau bij zowel het partiëel resistente als het vatbare genotype. De absolute verschillen tussen het vatbare en het resistente genotype voor bepaalde eigenschappen zoals biomassa namen echter toe naarmate planten ouder waren in de experimenten IV en V waarbij de luizen toegestaan was zich op alle delen (bladeren en stengel) van de planten te vestigen. Op basis van deze toegenomen absolute verschillen tussen resistente en vatbare planten is het dus beter om bij oudere planten (bijv. 30–40 dagen oud) op resistentie te selecteren.