Clonal populations of Clavibacter michiganensis subsp. michiganensis are responsible for the outbreaks of bacterial canker in greenhouse tomatoes in Italy

Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected in greenhouses from 17 farms during tomato bacterial canker outbreaks occurring between 2005 and 2008 in Sicily, were analysed by a multiphasic approach. Population studies were conducted to investigate the possible sources of inocula. Cmm strains were characterized by PCR assays targeting virulence genes, fingerprinting techniques, metabolic profiles and virulence. These strains were comparatively analysed with Cmm strains isolated in other parts of Italy over a period of 15 years. Chromosomal genes encoding virulence determinants tomA, ppaA, chpC, and the plasmid‐encoded genes pat‐1 and celA were detected by PCR in all tested strains, except for four Sicilian Cmm strains where the pat‐1 gene was not amplified. Using BOX‐PCR, Cmm strains were differentiated into 13 haplotypes and clonal populations were identified. Cmm strains isolated from different farms in 2008 showed the same BOX‐PCR haplotype. A distinct BOX‐PCR haplotype was obtained from atypical Cmm strains lacking pat‐1 and isolated in 2006/7 from three farms. Cmm strains with two different haplotypes were detected in one farm, whereas the other farms contained strains with only a single haplotype. A new fAFLP protocol based on the amplification of ApaI/MseI fragments was developed and was able to differentiate C. michiganensis subspecies. Different populations were delineated for the multiple outbreaks occurring in Sicily, whereas similar populations were recorded in other Italian regions over a period of 12 years. The results are consistent with previous studies that demonstrate that Cmm outbreaks are associated with propagation material.     

Survival of Clavibacter michiganensis subsp. michiganensis in tomato debris under greenhouse conditions

Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis, is one of the most important diseases of tomato worldwide. Once the pathogen has been introduced into an area, i.e. by contaminated seeds or transplants, it survives mainly on host debris. In different geographic areas the survival time of the pathogen in crop residues under field conditions has been very variable, ranging from 2 months in Morocco to 2 years in Iowa (USA). This study took place in the horticultural belt of Buenos Aires – La Plata, Argentina, where greenhouse production prevails, and monoculture with two production cycles per year is a common practice. The aim was to determine the survival time of this pathogen in plant residues left on the soil surface or buried. During three consecutive years, by the end of both production cycles in July (winter) and December (summer), above‐ (stem, petiole) and belowground (root) tissues were placed into nylon netting bags and left on the soil surface or buried at 10 cm depth. The pathogen population was regularly quantified by dilution plating on semiselective medium. In host debris left on the soil surface, bacteria survived 120–260 days for crop production cycles that ended in winter and 45–75 days for those that ended in summer. In stems or roots buried in winter, this period was 45–75 days. It is concluded that host debris, including roots, might be an important primary inoculum source of the pathogen in greenhouses.     

Bacteria from the citrus phylloplane can disrupt cell–cell signalling in Xanthomonas citri and reduce citrus canker disease severity

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a disease that affects almost all types of citrus crops. Production of particular Xcc pathogenicity factors is controlled by a gene cluster rpf, which encodes elements of a cell–cell communication system called quorum sensing (QS), mediated by molecules of the diffusible signal factor (DSF) family. Interference with cell–cell signalling, also termed quorum quenching, either by signal degradation or over‐production, has been suggested as a strategy to control bacterial disease. In this study, three bacterial strains were isolated from citrus leaves that displayed the ability to disrupt QS signalling in Xcc. Pathogenicity assays in sweet orange (Citrus sinensis) showed that bacteria of the genera Pseudomonas and Bacillus also have a strong ability to reduce the severity of citrus canker disease. These effects were associated with alteration in bacterial attachment and biofilm formation, factors that are known to contribute to Xcc virulence. These quorum‐quenching bacteria may represent a highly valuable tool in the process of biological control and offer an alternative to the traditional copper treatment currently used to treat citrus canker disease.     

拮抗菌Z-L-22不同剂型对番茄溃疡病的防治效果

以对番茄溃疡病菌拮抗效果良好的西唐链霉菌(Streptomyces setonii)菌株Z-L-22为研究对象,开发出3种大田应用剂型——水剂、颗粒剂和片剂,确定了片剂最佳发酵组分,并测定不同剂型在温室和棚室对番茄溃疡病的防治效果。温室试验结果表明,10×发酵液稀释液、固体发酵物5g和片剂1g于番茄定植时施用,对番茄溃疡病防效可达到80%以上;棚室番茄定植75d后,经过水剂、颗粒剂、片剂处理后的番茄发病率和严重度均显著低于硫酸链霉素处理和空白对照。其中片剂由于具有成本低、易储存运输、持效期长等优点,有很高的开发价值。     

不同药剂处理种子对番茄细菌性溃疡病菌的除害效果

利用不同剂量的磷酸三钠、次氯酸钙、苏纳米、甲醛、高锰酸钾、盐酸和二氯异氰尿酸钠溶液对带番茄细菌性溃疡病菌的番茄种子进行不同时间的处理,旨在筛选出各药剂除菌的最适处理剂量和最宜处理时间。借助传统PCR方法检测药剂处理对人工接菌种子的除害效果,同时采用活菌培养法测定各药剂对番茄细菌性溃疡病菌的抑制作用。结果显示,各药剂最适宜的除害处理条件为:8%磷酸三钠处理30 min,1%次氯酸钙处理20 min,0.4%苏纳米处理20 min,甲醛100倍液处理20 min,p H值1.5的盐酸处理10 min,1%高锰酸钾处理30 min,二氯异氰尿酸钠700倍液处理40 min。应用纸床法进行发芽试验,检测各药剂处理对番茄种子发芽的影响,结果表明,各药剂处理对番茄种子的发芽率没有明显不利影响。综合7种药剂的特点、最适除害处理条件和对发芽率的影响,二氯异氰尿酸钠为最理想的种子除害处理药剂。     

Genetics and ecology of the Entoleuca mammata-Populus pathosystem: Implications for aspen improvement and management

Quaking aspen is damaged and killed by many pathogens but throughout most of its range Entoleuca mammata, the cause of Hypoxylon canker is the most serious. Hypoxylon canker has been the subject of study for over 85 years, yet gaps in our understanding of this disease remain and practical control measures for existing stands are lacking. Numerous interacting host and pathogen factors have complicated investigations and have resulted in conflicting results among researchers. This synthesis of the literature examines our knowledge of the genetics of the host and pathogen and the biology of the disease. Regenerating dense stands and selecting superior, disease resistant clones with pre-infection resistance mechanisms based on long-term field trials are strategies most likely to be effective in minimizing losses caused by this disease.     

抗PthA-NLS多肽单克隆抗体的制备及单链抗体基因的构建

 【目的】制备抗PthA-NLS多肽单克隆抗体并构建其单链抗体（single chain variable fragment,ScFv）基因,为从PthA角度进一步研究柑橘溃疡病致病机理创造条件,并为构建单链抗体基因植物表达载体,尝试利用转基因和植物抗体技术创制抗溃疡病柑橘新种质奠定基础。【方法】利用PthA-NLS重组多肽免疫Balb/c小鼠,制备抗PthA-NLS多肽单克隆抗体,从分泌抗PthA-NLS多肽单克隆抗体的杂交瘤细胞中提取总RNA,采用RT-PCR和重叠延伸PCR（splicing by overlap extension PCR,SOE-PCR）法构建其单链抗体基因,并克隆到pGEM-T载体和原核表达载体pET32a(+)中,重组原核表达质粒转化大肠杆菌BL21(DE3)后诱导重组蛋白的表达。【结果】获得了3株能稳定分泌抗PthA-NLS多肽的单克隆抗体杂交瘤细胞株1C8H1、2D12B6、3D8A10。成功构建了单链抗体基因ScFv,获得的ScFv基因大小约750 bp,其中重链可变区（VH）基因有360 bp,轻链可变区（VL）基因有342 bp,中间连接肽基因45 bp,经过IPTG诱导ScFv原核表达,获得了大小为44.5 kD的ScFv重组蛋白。【结论】试验获得了3株能稳定分泌抗PthA-NLS多肽的单克隆抗体杂交瘤细胞株,成功构建了ScFv基因,并实现了原核表达,为进一步探索PthA的致病机理和下一步利用抗体技术获得抗溃疡病柑橘种质的研究打下了基础。     

山核桃干腐病发生发展规律及防治技术

山核桃干腐病Macrophoma caryae是山核桃Carya cathayensis林的一种新病害。研究发现,该病每年3月下旬始发,至11月下旬以菌丝体在病组织越冬。为了有效控制该病的蔓延,选择14种药剂对它们开展单稀释倍数抑菌试验,并将抑菌效果好的3种药剂进行8种梯度稀释倍数试验。再按筛选出的最佳防治药剂及其稀释倍数进行实地防治试验,获得最佳防治药荆和方法。结果表明：当4—5月山核桃干腐病的病原菌孢子发生盛期,在病株上喷洒体积分数为80％402抗菌剂乳油1：100～1：2500倍液、80％乙蒜素乳油1：100～1：2500倍液和95％硫酸铜晶体1：100～1：500倍液的可选配比溶液时,均能有效控制山核桃干腐病病原菌孢子的蔓延：而在8—9月该病病原菌入侵山核桃木质部时,在刮除病斑或在病斑上深划线后,再用80％乙蒜素乳油、80％402抗菌剂和95％硫酸铜晶体中的任何一种杀菌剂的1：100～1：500倍溶液进行喷雾防治,15d后均可见明显的防治效果。图1表3参9     

新疆核桃树腐烂病致病力室内测定方法的研究

为探索出快速、可靠及操作简便的新疆核桃树腐烂病室内致病力测定方法,通过不同伤口类型将强致病力菌株的菌饼、分生孢子悬浮液和菌悬液接种在‘温185’核桃品种3种枝龄的枝条上,观测4种不同温度处理后核桃树腐烂病的发病程度得出最适致病条件。结果表明：3年生枝龄的核桃腐烂病发病最严重,病斑长度达到8.5 cm;通过烫伤处理后利用菌饼接种的离体枝条病斑长度最大,为9.8 cm;最利于病原菌扩展的温度为25℃,病斑长度达到11 cm;烫伤后放置0、1、2、3、4天后接种,病斑长度扩展无显著性差异性。研究结果为核桃腐烂病室内致病力的快速测定提供理论依据。     

Symptom development of the resinous stem canker caused by inoculation withCistella japonica ontoChamaecyparis obtusa

Cistella japonica was inoculated onto the stems of youngChamaecyparis obtusa trees, and the development of external and internal symptoms was investigated for five years. Most lesions started exuding resin from May to July during the first growing season after inoculation, while a few lesions started exuding resin during the second growing season. Resin exudation lasted for two successive years on most lesions, and during successive three, four, or five years on some lesions. Resin exuded excessively in the secondary phloem of the lesions and resin cysts frequently developed there. The lesions where cambial tissue was necrosed occupied 18% of all the lesions and the resinous areas expanded to a larger size than those where resin simply exuded in the phloem.Ci. japonica was reisolated from some inoculated lesion tissues at a high frequency. The isolation frequency of the fungus from some lesion tissues two and more years later was zero or low and that ofCryptosporiopsis abietina was high. This work was supported by a grant from the Forest Agency of the Japanese Government.