Multilocus sequence typing analysis of Italian Xanthomonas campestris pv. campestris strains suggests the evolution of local endemic populations of the pathogen and does not correlate with race distribution

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in Brassicaceae. It is widespread in Italy and severe outbreaks occur under conditions that favour disease development. In this study a multilocus sequence typing approach (MLST) based on the partial sequence of seven loci was applied to a selection of strains representative of the main areas of cultivation and hosts. The aim was to investigate whether the long tradition of brassica crops in Italy has influenced the evolution of different Xcc populations. All loci were polymorphic; 14 allelic profiles were identified of which 13 were unique to Italian strains. Based on the seven loci, the most common genotype within the Italian Xcc strains (AP1) was also the most representative genotype found in worldwide Xcc strains. This genotype was included in a new clonal complex in addition to three other clonal complexes already identified in Xcc populations. The phylogenetic reconstruction using a concatenated dataset of four conserved protein-coding genes, dnaK, fuyA, gyrB and rpoD, showed that the Italian strains belonged to two genetic groups. Physiological races were also investigated for the first time in Italy. The race structure of Xcc was determined by inoculating eight differential Brassica lines belonging to five species and showed that, in Italy, race 4 is the most widespread, followed by races 1 and 6. No correlation was found between allelic profiles, host of isolation, geographical origin and races, although a prevalent race was identified within the same clonal complex.     

Trichoderma species associated with green mould disease of Pleurotus ostreatus and their sensitivity to prochloraz

Green mould disease causes serious economic losses in Pleurotus ostreatus crops worldwide, including in Italy, where prochloraz is the only chemical fungicide allowed to control the disease. The effectiveness of the doses 0.01, 0.05, 0.25 and 1.25 μL L⁻¹ (field dose) of prochloraz (Sponix Flow, 450 g L⁻¹), against colony growth rate and spore germination of Trichoderma pleuroti, T. pleuroticola and T. guizhouense strains on wheat straw extract agar plates were evaluated. Complete inhibition of Trichoderma pleuroti and T. pleuroticola growth was shown by the field dose of prochloraz, and also by the 0.25 μL L⁻¹ dose for T. pleuroti. Complete inhibition of spore germination occurred for all Trichoderma strains at field dose, and at 0.25 μL L⁻¹ for T. pleuroti strains. In in vivo assays, the effect of prochloraz doses 0.05, 0.25 and 1.25 μL L⁻¹ on colonization of straw substrate by T. pleuroti, T. pleuroticola and T. guizhouense inoculated at two spore densities (10² and 10⁵ spores mL⁻¹) immediately after P. ostreatus spawn was studied. Trichoderma pleuroti and T. pleuroticola were both responsible for green mould disease, whereas T. guizhouense was not pathogenic. Trichoderma pleuroti was more aggressive than T. pleuroticola. Prochloraz was effective against T. pleuroti at the field dose, and against T. pleuroticola at 0.25 and 1.25 μL L⁻¹. The study on Trichoderma × Pleurotus interaction type showed that Trichoderma species were active against the mycelial growth of P. ostreatus by competition for space and nutrients, and neither hyphal interaction nor effect by volatile or nonvolatile metabolites occurred.     

Cryptic species and population genetic structure of Plasmopara viticola in São Paulo State,Brazil

Downy mildew (Plasmopara viticola) is one of the most important diseases in grape-growing areas worldwide, including Brazil. To examine pathogen population biology and structure, P. viticola was sampled during the 2015/16 growing season from 516 lesions on nine grape cultivars in 11 locations in subtropical areas of São Paulo State, Brazil. For identification of cryptic species, a subsample of 130 isolates was subjected to cleaved amplified polymorphic sequence (CAPS) analysis, and for 91 of these isolates the ITS1 region was sequenced. These analyses suggest that the population of P. viticola in São Paulo State consists of a single cryptic species, P. viticola clade aestivalis. Seven microsatellite markers were used to determine the genetic structure of all 516 P. viticola isolates, identifying 23 alleles and 55 multilocus genotypes (MLGs). Among these MLGs, 34.5% were clonal and represented 93% of the isolates sampled. Four dominant genotypes were present in at least five different locations, corresponding to 65.7% of the isolates sampled. Genotypic diversity (Ĝ = 0.21–0.89) and clonal fraction (0.58–0.96) varied among locations (populations). Most populations showed significant deviation from Hardy–Weinberg expectations; in addition, excess of heterozygosity was verified for many loci. However, principal coordinate analysis revealed no clusters among locations and no significant isolation by distance was found, suggesting high levels of migration. The results indicate that downy mildew epidemics result from multiple clonal infections caused by a few genotypes of P. viticola, and reproduction of P. viticola in São Paulo State is predominantly asexual.     

Exogenous application of melatonin improves plant resistance to virus infection

In this study, melatonin (MEL)-mediated plant resistance to tobacco mosaic virus (TMV) was examined to study local infection in Nicotiana glutinosa and systemic infection in Solanum lycopersicum. Exogenous application of 100 µm MEL increased anti-virus infection activity to 37.4% in virus-infected N. glutinosa plants. The same treatment significantly reduced relative levels of virus RNA analysed by qRT-PCR and virus titres measured by dot-ELISA, and increased the relative expression levels of the PR1 and PR5 genes analysed by qRT-PCR, in virus-infected S. lycopersicum. MEL treatment induced considerable accumulations of salicylic acid (SA) and nitric oxide (NO) but did not significantly affect production of hydrogen peroxide (H₂O₂) in the virus-infected S. lycopersicum plants. Transgenic nahG N. tabacum was used to determine whether MEL-induced TMV resistance was dependent on the SA pathway. The results showed that the relative RNA level of the TMV analysed by qRT-PCR and virus titres analysed by dot-ELISA were not reduced by the MEL treatment in the nahG transgenic N. tabacum seedlings treated twice with 100 µm MEL. The increased relative expression levels of PR1 and PR5 were greatly reduced when cPTIO, an NO scavenger, was included in the MEL treatment. A working model of MEL-mediated plant resistance to TMV is proposed. MEL-mediated plant resistance to viruses provides a new avenue to control plant viral diseases.     

Phytophthora oleae,a new root pathogen of wild olives

Wild olive (Olea europaea subsp. europaea var. sylvestris) is an important component of Mediterranean forests and a key genetic source for olive improvement programmes. Since 2009, a severe decline caused by Phytophthora cryptogea and P. megasperma has been detected in a protected wild olive forest of high ecological value (Dehesa de Abajo, Seville, Spain). In this natural forest, sampling of roots and soil was carried out on 25 wild olives with symptoms in 2014 and 2015. Apart from the already known P. cryptogea A1 and P. megasperma, a third Phytophthora species was consistently isolated from wild olive rootlets with symptoms. These isolates conformed morphologically with the newly described species P. oleae and were confirmed by analysis of their ITS regions and cox1 sequences. Temperature–growth relationships showed a maximum growth at 19.9 °C on carrot agar medium, making it the lowest temperature Phytophthora species infecting wild olive roots. Pathogenicity was confirmed on 1-year-old healthy wild olive seedlings and was similar to the previously known pathogenic phytophthoras. As temperature requirements are quite different, the three Phytophthora species may be active against wild olive roots in different seasons. However, the prevalence of P. oleae infecting wild olives in recent years could be due to its introduction as a new invasive pathogen. The probable invasive nature of P. oleae, together with increasing rain episodes concentrated in short periods frequent in southern Spain, would allow the outbreak of infections in wild olive forests, and also put cultivated olive orchards at risk.     

寄主植物中抑制十字花科黑腐病菌Ⅲ型分泌系统小分子化合物的鉴定

 寻找抑制植物病原菌III型分泌系统的植物源活性小分子化合物,是研发生物安全农药的重要途径之一。本研究采用水煮提取法从十字花科黑腐病菌寄主植物满身红萝卜中提取分离活性小分子化合物,利用高效液相-质谱联用解析出活性物质的单体结构。然后用荧光素酶基因luxAB构建融合报告系统以及定量PCR检测活性物质对十字花科黑腐病菌III型分泌系统的抑制效果,最后采用剪叶接种和压渗接种的方法研究活性小分子物质对十字花科黑腐病菌的生防作用。研究表明,植物鞘氨醇和二氢鞘氨醇对十字花科黑腐病菌III型分泌系统基因的转录表达有一定程度的抑制作用,但是对I、II、IV型分泌系统基因的表达没有明显的抑制作用。植物鞘氨醇在XCM1上影响菌的生长,而二氢鞘氨醇不影响菌的生长。同时,还发现这两种物质能显著降低Xcc在寄主植株满身红萝卜上的病害症状以及能够使Xcc在非寄主植物辣椒上引起过敏反应的能力丧失。该研究结果为深入研究小分子化合物对十字花科黑腐病菌III型分泌系统的作用机制及后续开发植物源抑制剂提供了一定的理论依据。     

稻瘟病菌F-box蛋白MoFbr7生物学功能分析

F-box蛋白在泛素-蛋白酶体途径(ubiquitin-proteasome pathway,UPP)中参与调控胞内蛋白降解、受体识别、信号传导等生物学过程。本研究从稻瘟病菌中克隆了F-box基因MoFbr7,序列分析表明,该基因编码产物具有1个F-box结构域(N-端)和8个连续的WD40重复序列(C-端),在丝状真菌中高度保守。利用基因敲除方法,获得4个MoFbr7基因敲除突变体,同时构建了回补菌株。表型分析结果显示,MoFbr7基因缺失突变体在产孢量、附着胞形态、原生质体释放、致病性等方面均无异常。突变体在MM、RDC培养基上生长速率下降;对细胞壁胁迫因子CFW(Calcofluor white)、刚果红敏感。以上结果表明MoFbr7参与稻瘟病菌的营养生长与细胞壁完整性,为进一步揭示其生物学功能奠定基础。     

棉铃虫脂肪酸结合蛋白的克隆与表达

为深入了解棉铃虫Helicoverpa armigera脂肪酸结合蛋白HaFABP1在参与细胞内脂肪酸代谢过程中的功能,基于酵母单杂交结果从幼虫中肠cDNA中扩增得到HaFABP1的开发阅读框,构建重组菌株BL21-pET28a-HaFABP1,进行融合蛋白His-HaFABP1的诱导和纯化,并利用实时荧光定量PCR检测棉铃虫不同时期和组织中HaFABP1的表达规律,最后检测2-十三烷酮处理下棉铃虫6龄幼虫中肠组织内HaFABP1的变化情况。结果显示,克隆获得的棉铃虫HaFABP1为405 bp,编码134个氨基酸,相对分子量和等电点分别为15.08 kD和5.54;在37℃下用0.5 mmol/L异丙基硫代半乳糖苷(IPTG)诱导重组菌株4 h后,得到约18.4 kD的可溶性蛋白His-HaFABP1,纯化后的融合蛋白条带单一且大小符合预期;HaFABP1在棉铃虫的脂肪体、中肠、体壁和头部中都有表达,在头部的表达量最低,在中肠中的表达量最高;HaFABP1在棉铃虫幼虫的每个发育时期都有表达,表达量总体呈现先降低后升高的趋势,4龄时最低而1龄时最高;10 mg/g 2-十三烷酮处理饲料饲喂棉铃虫6龄幼虫6 h和15 h后,中肠内HaFABP1的表达量与对照相比显著提高,且随着时间的延长逐渐增加。表明HaFABP1与棉铃虫的生长发育有关,并且有可能参与了昆虫的解毒过程。     

温度胁迫下沙葱萤叶甲小热激蛋白基因GdHsp20.6的克隆及表达分析

为明确小热激蛋白在沙葱萤叶甲Galeruca daurica应对温度胁迫中的作用,采用PCR方法克隆沙葱萤叶甲小热激蛋白基因Hsp20（GdHsp20.6）完整的开放阅读框（open reading frame,ORF）序列,应用在线软件对GdHsp20.6基因进行生物信息学分析,通过原核表达技术诱导表达及纯化其编码蛋白,并通过实时荧光定量PCR（real-time quantitative PCR,RT-qPCR）技术分析不同温度胁迫下GdHsp20.6基因的表达量。结果显示,GdHsp20.6基因ORF序列长度为543 bp,编码180个氨基酸,预测分子量为20.6 kD,无跨膜区和信号肽。GdHsp20.6氨基酸序列有高度保守的α-结构域。GdHsp20.6氨基酸序列与其它鞘翅目昆虫的Hsp20氨基酸序列有较高的一致性,其中与花绒寄甲Dastarcus helophoroides的Hsp20.99氨基酸序列的一致性最高,为63%。GdHsp20.6基因在大肠杆菌Escherichia coli BL21（DE3）细胞系中成功表达,经异丙基-β-D-硫代吡喃半乳糖苷（isopropyl-β-d-thiogalactoside,IPTG）诱导后GdHsp20.6蛋白成功表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）和Western-blot分析表明融合蛋白大小与预测大小一致,并纯化获得了纯度较高的目的蛋白GdHsp20.6。低温（-10~5℃）和高温（35~40℃）处理1 h以及处理后25℃恢复30 min均能诱导GdHsp20.6基因表达上调,并且0℃处理30~120 min也能诱导GdHsp20.6基因表达上调。表明GdHsp20.6基因在沙葱萤叶甲应对低温和高温胁迫中可能起着重要作用。     

韭菜迟眼蕈蚊紫外敏感视蛋白基因的克隆及光强度对其表达量影响

为明确韭菜迟眼蕈蚊Bradysia odoriphaga紫外敏感视蛋白基因Bo-uv的作用及其与趋光性的关系,利用常规PCR方法克隆获得Bo-uv基因的全长cDNA序列,分析了其敏感视蛋白的氨基酸序列与其它12种昆虫同源蛋白氨基酸序列之间的系统进化关系,运用qPCR技术检测了不同发育阶段、不同组织及不同光强度下Bo-uv基因的相对表达量。结果表明,Bo-uv基因cDNA全长2 757 bp,开放阅读框1 542 bp,编码514个氨基酸。韭菜迟眼蕈蚊紫外敏感视蛋白的氨基酸序列与其它12种昆虫同源蛋白的氨基酸序列一致性为21.93%～43.00%,与橘小实蝇Bactrocera dorsalis的氨基酸序列同源性最高。Bo-uv基因在韭菜迟眼蕈蚊蛹末期、成虫期表达,在成虫头部的相对表达量较高。在0～10 000 lx光强范围内雌、雄成虫体内该基因的相对表达量均呈先增高后降低趋势。与对照相比,1 000 lx光强度下其相对表达量显著升高,10 000 lx时相对表达量显著降低。表明光强度能够有效地调控Bo-uv基因的表达,该基因在韭菜迟眼蕈蚊感知外界光刺激过程中具有重要作用。