Resistance and Susceptibility of Arabidopsis thaliana to Bacterial Wilt Caused by Ralstonia solanacearum

ABSTRACT Tomato bacterial wilt caused by Ralstonia solanacearum is a model system for studying plant-bacterial interactions, because it is genetically one of the best characterized plant diseases. We demonstrate here that four different strains of R. solanacearum, two from radishes (Rd4 and Rd15) and two from tomato (Ps21 and Ps95), can infect 27 different ecotypes of Arabidopsis thaliana, causing different responses. All ecotypes tested were highly susceptible to strain Rd15, which caused symptoms similar to those observed in tomato plants. For example, leaf drooping and discoloration developed just 3 days after inoculation, and plants completely wilted within 1 week. Strains Rd4 and Ps95 were less infectious than Rd15. With these two strains, a variety of disease responses were observed among different ecotypes at 2 weeks after inoculation; both susceptible and resistant ecotypes of A. thaliana were identified. Ps21 was the least infectious of the four strains and caused almost no symptoms in any of the ecotypes of Arabidopsis tested. Direct bacterial isolation and plant skeleton hybridization analysis from infected plants indicated that bacterial colonization was correlated with the severity of symptoms. Growth of bacteria was limited to the infection site in resistant plants, whereas the bacteria spread throughout susceptible plants by 1 week after inoculation.     

A Single Locus Leads to Resistance of Arabidopsis thaliana to Bacterial Wilt Caused by Ralstonia solanacearum Through a Hypersensitive-like Response

ABSTRACT Strains of Ralstonia solanacearum have been shown to cause bacterial wilt in some, but not all, ecotypes of Arabidopsis thaliana. We demonstrate here that after inoculation of the leaves of resistant ecotype S96 with R. solanacearum strain Ps95 necrosis around the inoculation site rapidly appeared and no further symptoms developed in the plant. Leaves of susceptible ecotype N913 completely wilted 7 days after inoculation with Ps95, and symptoms spread systemically throughout the whole plant within 2 weeks after inoculation. These results suggest that the resistance of Arabidopsis S96 to R. solanacearum is due to a response similar to the hypersensitive response (HR) observed in other plant diseases. Northern blot analysis of the expression of defense-related genes, known to be differentially induced during the HR in Arabidopsis, indicated that pathogenesis-related protein PR-1, glutathione S-transferase (GST1), and Cu, Zn superoxide dismutase (SOD) mRNAs increased significantly in S96 leaves between 3 to 12 h after infiltration with Ps95. The induction of these genes in susceptible ecotype N913 by Ps95 was clearly delayed. Genetic analysis of crosses between resistant ecotype S96 and susceptible ecotype N913 indicated that resistance to Ps95 is due to a single dominant locus.     

防治青枯病工程菌Hrp-菌株的发酵培养基配方优化

在摇瓶发酵条件下,采用全因子实验设计法和正交实验设计法对工程菌Hrp-菌株的培养基碳、氮源配方进行优化,确定了最适碳源为葡萄糖、细玉米面,最适氮源为硫酸铵、细豆饼粉;对这四个碳氮源因子的浓度进行L9（34）的正交试验,最终确定出该菌的最适发酵培养基碳、氮源配方为（g/L）：葡萄糖8g、细玉米面5g、细豆饼粉35g、硫酸铵2g。优化后的培养基配方使摇瓶发酵液菌量从1.34×10^10cfu/ml提高至3.30×10^10cfu/ml。     

繁殖青枯菌噬菌体无毒菌株的筛选及应用

利用噬菌体防治植物细菌性病害,关键技术是噬菌体的扩大繁殖,利用宿主菌繁殖,应用时存在一定的风险。本研究通过继代培养筛选致病性丧失的青枯菌菌株作为青枯菌噬菌体扩繁的宿主,结果表明,通过对野生青枯菌株RSsw326连续超过20代的培养,获得了一株菌落圆形、游动性弱的变异菌株;再通过刺叶、注射和伤根等方法接种烟苗,30 d后无萎蔫症状出现,将该菌株命名为RSsw326-2;测试表明,该菌株对青枯病菌没有拮抗作用,可被从福建不同烟区分离纯化的8株噬菌体裂解。利用菌株RSsw326-2作为宿主,进行青枯菌噬菌体的扩大繁殖,培养36 h,效价可达10¹⁰ PFU/mL。将无毒菌株RSsw326-2+噬菌体的共培养液刺叶、伤根和不伤根接种烟苗,35 d后均无症状出现,而将毒性菌株RSsw326+噬菌体的共培养液刺叶接种后14 d、伤根接种后28 d发病率都为100%,不伤根接种烟苗35 d,发病率为66.7%。无毒菌株RSsw326-2+噬菌体的共培养液对盆栽烟苗防病试验结果表明,发病时间推迟8 d,并且在接种后21 d时对烟草青枯病的防效达74.1%,显著高于对照组。本研究采用继代培养筛选获得的无毒菌株作为噬菌体扩大繁殖的宿主,为今后研制噬菌体制剂的生产提供了参考。     

抗姜青枯菌银杏内生真菌的分离及抑菌特性

为了筛选新的生防菌种资源,从银杏根、叶中分离出对青枯劳尔氏菌Ralstonia solanacearum有拮抗作用的内生真菌,结合形态特征和ITS序列对该菌株进行了鉴定,分析了菌株发酵液的抑菌特性。结果表明,分离获得的80株内生真菌中有5株对青枯菌有拮抗作用,其中Gbh45具有良好的抑菌效果和遗传稳定性,根据形态特征和ITS序列比对,该菌株被鉴定为球黑孢菌Nigrospora sphaerica。该菌株的发酵产物经紫外照射、不同温度处理后其抑菌活性不发生变化,在碱性条件的耐受能力高于酸性,表明菌株Gbh45的发酵产物在自然条件下稳定性高,该菌对姜青枯病的生物防治具有巨大的应用潜力。     

两株植物根际促生菌对番茄青枯病的生物防治效果评价

研究了Erwinia persicinus RA2和Bacillus pumilus WP8浸种和拌土处理对番茄青枯病的实际防治效果,及其对番茄根际微生物群落的影响。结果显示,两株菌都具有防治番茄青枯病的作用,并能不同程度地促进番茄幼苗生长。主要体现在显著提高番茄幼苗健株率,病原菌处理的健株率最低,仅为22.4%,而RA2和WP8浸种处理分别达68.9%和62.8%;促进幼苗地上部增高、增粗和根部生长,如WP8浸种处理的茎叶干重和根干重分别达到4.87 mg·株^-1和35.69 mg·株^-1,分别比病原菌对照提高110.82%和205.83%。浸种处理的促进效应明显优于拌土处理;还能在一定程度上提高土壤水稳性团聚体（〉0.25 mm）比例,WP8浸种处理尤为明显,分别比空白对照和病原菌对照提高269.91%和156.88%。DGGE指纹图谱表明根际微生物群落受番茄种植的影响最大,其次是青枯病菌,而受这两种菌施用的影响最小。     

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.     

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.     

苏云金芽胞杆菌杀虫晶体蛋白Cry1D新基因的克隆与特性

为扩充鳞翅目害虫杀虫基因资源,本研究从苏云金芽胞杆菌BN23-5中克隆得到一个新的cry基因,并对其进行鉴定和分析。该基因为一个完整的cry1D基因,全长3501 bp,编码1166个氨基酸残基。该氨基酸序列是一个新的Cry氨基酸序列,与Cry1Db1的同源性最高,为86%,命名为Cry1Dd1（登录号为KJ728844）。将该基因插入穿梭表达载体pSTK中,转入BT无晶体突变株HD73^-中进行表达。结果表明,cry1Dd1基因能在BT无晶体突变株中表达,并形成菱形伴孢晶体。SDS-PAGE验证其分子量为132.2 kD,与预测的大小相符。生物活性测定表明,Cry1Dd1晶体蛋白对小菜蛾的幼虫具有杀虫活性,LC₅₀为13.1 μg/mL;能明显抑制甜菜夜蛾幼虫的生长;但对棉铃虫幼虫没有杀虫活性。对cry1Dd1基因序列进行分析,cry1Dd1包含8个block保守区域,这和目前其他的cry基因相似;Cry1Dd1蛋白的活性区域为N端的37~593位氨基酸残基。     

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.     

Isolation and Characterization of a Cold-Tolerant Strain of Fusarium proliferatum, a Biocontrol Agent of Grape Downy Mildew

ABSTRACT A cold-tolerant strain of the mycoparasite Fusarium proliferatum was isolated following UV mutagenesis of the G6 strain, which is a biocontrol agent of grape downy mildew. The isolated strain (designated 1505) exhibited radial growth two to threefold that of the parent strain when grown at 13 degrees C, which is generally suboptimal for growth of Fusarium spp., but desirable for its host, Plasmopara viticola. This rapid growth was correlated with improved biological control of P. viticola, determined by a detached-leaf assay. Even though radial growth of strain 1505 at higher temperatures was slower than that of G6 and the strain failed to conidiate, there was no reduction in biocontrol efficacy. Significantly higher levels of extracellular beta-glucosidase and endo-1,4-beta-glucanase activity were measured in the culture filtrate of strain 1505 relative to that of strain G6. A DNA-mediated transformation procedure that included the introduction of antibiotic resistance and a GUS reporter gene system was adapted for F. proliferatum. Using the GUS-engineered strains, we demonstrated that both G6 and 1505 exhibit the characteristic coiling and penetration of host structures.     

Genetic Marker Analysis of a Global Collection of Isolates of Citrus tristeza virus: Characterization and Distribution of CTV Genotypes and Association with Symptoms

ABSTRACT Genetic markers amplified from three noncontiguous regions by sequence specific primers designed from the partial or complete genome sequences of Citrus tristeza virus (CTV) isolates T3, T30, T36, and VT were used to assess genetic relatedness of 372 isolates in an international collection. Eighty-five isolates were judged similar to the T3 isolate, 81 to T30, 11 to T36, and 89 to VT. Fifty-one isolates were mixed infections by two or more identifiable viral genotypes, and 55 isolates could not be assigned unequivocally to a group defined by marker patterns. Maximum parsimony analysis of aligned marker sequences supported the grouping of isolates on the basis of marker patterns only. Specific disease symptoms induced in select citrus host plants were shared across molecular groups, although symptoms were least severe among isolates grouped by markers with the T30 isolate and were most severe among isolates grouped by markers with the T3 isolate. Isolates assigned the same genotype showed variable symptoms and symptom severity. A classification strategy for CTV isolates is proposed that combines genetic marker patterns and nucleotide sequence data.     

Characterization of Two Major Genetic Factors Controlling Quantitative Resistance to Melampsora larici-populina Leaf Rust in Hybrid Poplars: Strain Specificity, Field Expression, Combined Effects, and Relationship with a Defeated Qualitative Resistance Gene

ABSTRACT Two genetic factors explain a significant proportion of the variability for quantitative resistance to Melampsora larici-populina leaf rust in a Populus deltoides x P. trichocarpa F(1) progeny. One is inherited from P. deltoides and is associated with a defeated qualitative resistance gene R(1), and the other, R(US), is inherited from P. trichocarpa. To assess the potential contribution of these two factors for durable resistance breeding, 284 genotypes from this F(1) progeny were studied in laboratory experiments with three M. larici-populina strains and in a field experiment under natural inoculum pressure. Results confirmed that both factors can have strong beneficial effects in the laboratory. These effects were strain specific, thus impairing their chances for durability. However, association of both factors led to synergistic effects in most situations. In accordance with good field-laboratory relationships, especially those involving uredinia-size laboratory measurements, field effects of both resistance factors were significant. R(US) led to a significant reduction of rust colonization on the most infected leaf in the field, and its effect was significant both in the presence and the absence of R(1). In contrast, the presence of R(1) was useful in the field only when R(US) was absent. The nature of the genetic relationship between both factors remains unknown, but benefits from their association should be quantified over a longer period to evaluate potential adaptation of the pathogen.     

Euphresco inter‐laboratory comparison (2009–2012) on detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers: proposal to include TaqMan® real‐time PCR as a primary (core) screening test in EU/EPPO standard methods

In the European Union (EU) potato production is surveyed for Clavibacter michiganensis subsp. sepedonicus (potato ring rot) and Ralstonia solanacearum (potato brown rot) under Commission Directives 93/85/EEC with its amendment 2006/56/EC and 98/57/EEC with its amendment 2006/63/EC. A regular update of the Directives is required in view of developments in understanding of the biology of these organisms and the diagnostics recommended for their detection and identification. Three inter‐laboratory tests (ILT1, ILT2 and ILT3) were performed from 2009 to 2012 as part of a Euphresco Phytosanitary ERA‐NET project to assess performance of current official methods for C. michiganensis subsp. sepedonicus and R. solanacearum. A major aim of the ILTs was to generate data on the performance of real‐time PCR protocols to support their introduction as primary (core) screening tests for both pathogens. In ILT1, 29 laboratories from 23 countries participated, in ILT2, 23 laboratories from 18 countries and in ILT3 42 laboratories from 24 countries. Relative accuracies for real‐time PCR tests averaged 92% for R. solanacearum and 96% for C. michiganensis subsp. sepedonicus) and compared with existing primary (core) screening tests (immunofluorescence, conventional PCR, semi‐selective plating and bioassay) in terms of analytical sensitivity, analytical specificity and robustness. It was concluded that all methods tested, including real‐time PCR, can be considered as equivalent. Therefore TaqMan ^® real‐time PCR is recommended for inclusion in EU Directives and EPPO Standards as a reliable primary (core) screening method.