Molecular tracing of the transmission routes of bois noir in Mediterranean vineyards of Montenegro and experimental evidence for the epidemiological role of Vitex agnus‐castus (Lamiaceae) and associated Hyalesthes obsoletus (Cixiidae)

Epidemiological aspects and transmission routes of bois noir (BN), a grapevine yellows disease induced by ‘Candidatus Phytoplasma solani’, have been exhaustively studied in the affected vineyards of continental Europe but not in the Mediterranean coastal zone. Because ‘Ca. Phytoplasma solani’ and its principal vector Hyalesthes obsoletus presumably originate from the Mediterranean, gaining knowledge of the epidemiological peculiarities of the disease in this area is essential for understanding its global spread and diversification, as well as for designing local management strategies. In this study, molecular epidemiology was applied to trace transmission pathways of ‘Ca. Phytoplasma solani’ in the Mediterranean vineyards of Montenegro, using multilocus sequence typing of tuf, vmp1 and stamp genes of the isolates associated with various hosts. Thus, ‘Ca. Phytoplasma solani’ was tracked from a tentative reservoir plant (inoculum source) through an associated vector population to the infected grapevine. Three pathways of transmission were documented, originating from Urtica dioica, Convolvulus arvensis and Vitex agnus‐castus; however, only the route originating from U. dioica was direct, whereas the latter two were overlapping and could be intermixed. Vitex agnus‐castus is a natural source of ‘Ca. Phytoplasma solani’, representing an important link in disease epidemiology in the Mediterranean and a possible origin of several genotypes occurring in central Europe. Experimental confirmation of the role of Vitex‐associated H. obsoletus in BN transmission in Montenegrin vineyards indicates its tentative role as a vector in the wide area of the Mediterranean, where some of the major wine‐producing regions are located.     

Development of a specific assay using RISA for detection of the bacterial agent of 'basses richesses' syndrome of sugar beet and confirmation of a Pentastiridius sp. (Fulgoromopha, Cixiidae) as the economic vector

A technique for the specific diagnosis in insects of SBRp (the γ-3 proteobacterium associated with the syndrome 'basses richesses' (SBR) of sugar beet crops in eastern France), using the RISA (rDNA intergenic spacer analysis) technique, was developed. PCR using the Alb1/Oliv1 primer pair specifically amplified a 16S-ITS region of SBRp and produced a characteristic DNA fingerprint. This PCR assay did not detect other closely related organisms, including the Arsenophonus endosymbiont of Diaphorina citri , the secondary endosymbiont of Glycaspis brimblecombei , or ' Candidatus Phlomobacter fragariae', a related phytopathogenic γ-3 proteobacterium. Six different ribosomal operons, differing in their ITS region or partial 16S sequence, were identified in SBRp. PCR amplification with Alb1/Oliv1 of DNA samples from Hemiptera species (suborders Fulgoromorpha and Cicadomorpha) collected in sugar beet fields confirmed Pentastiridius sp. as the economic vector of SBR disease. The high percentage of field- Pentastiridius sp. specimens which tested positive for SBRp reflected the importance of SBR disease in sugar beet crops. This is the first time that the RISA technique has been used as a diagnostic test for a plant pathogenic bacterium in insects.     

Oncopsis alni (Schrank) (Auchenorrhyncha: Cicadellidae) as a Vector of the Alder Yellows Phytoplasma of Alnus glutinosa (L.) Gaertn.

Alder yellows phytoplasma was detected by PCR in Alnus glutinosa trees in the Palatine and Mosel areas of Germany. The restriction profiles obtained by TaqI and AluI digestion of a PCR amplified ribosomal DNA fragment from this phytoplasma and a periwinkle isolate of alder yellows from Italy (ALY) could not be distinguished while elm yellows isolates from Europe and North America led to different fragment patterns. Different restriction profiles for ALY and the German alder phytoplasma were obtained by TruI digestion of a non-ribosomal DNA fragment. Phloem feeding insects were collected from infected alder trees. Phytoplasmas of the elm-yellows group were detected by PCR in psyllids and the leafhopper Oncopsis alni. These pathogens were indistinguishable from the phytoplasma found in alder. Only O. alni was able to transmit the pathogen to healthy alder seedlings. Thus, it is the first insect known to transmit this phytoplasma. This leafhopper could be responsible for the ubiquitous infection of Alnus glutinosa due to its close association with alder and its wide distribution in Europe.     

Spatial and temporal stolbur population structure in a cv. Chardonnay vineyard according to vmp1 gene characterization

Bois noir is a grapevine disease caused by the stolbur phytoplasma. It is widespread in all European and Mediterranean viticultural areas, and it can induce severe damage to the quality and quantity of production. The recent disease recrudescence has encouraged studies on the use of molecular markers to assess the genetic diversity of stolbur strains. The aim of this study was to evaluate the presence of Bois noir symptoms and to monitor the spatial genetic structure of the stolbur population according to vmp1 genotypes, through 2011 and 2012 in a cv. Chardonnay vineyard. In both years, there were increased vines with symptoms from July to September. The analysis of dispersal indices showed that the spatial distribution was uniform in the vineyard. However, the two‐dimensional contour maps show that Bois noir severity was higher in plants located on the borders than in the central parts of the vineyard. Stolbur population was composed of two prevalent vmp genotypes (V14, V12) across both years, along with other minor haplotypes (V3, V4, V9, V11, V15, V18, in 2011; V3, V18 in 2012). The data indicate that the vmp1 gene is an efficient marker to study the population structure of stolbur phytoplasma, to track the movement of the pathogen, and to identify the inoculum source, which will all serve in the planning of control strategies.     

Macropsis mendax as a vector of elm yellows phytoplasma of Ulmus species

A 3-year study was carried out in north-east Italy, the site of recent elm yellows epidemics, to identify vectors for the elm yellows phytoplasma. Using PCR analysis, Ulmus minor and Ulmus pumila , each with and without symptoms, were positive for the elm yellows phytoplasma. Macropsis mendax , a univoltine and monophagous leafhopper, was shown to be the vector of the elm yellows-associated disease agent. PCR analyses demonstrated that the insect was infected both in natural conditions and in the screenhouse after acquisition-feeding on infected elm plants. Groups of M. mendax , collected from naturally infected elm trees, transmitted elm yellows phytoplasma to elm test plants. In nature, Alnus glutinosa trees affected by alder yellows were found in the surroundings of yellows-affected elm trees; the associated disease agent of alder yellows was transmitted under controlled conditions from alder to elm test plants by grafting.     

Molecular characteristics of phytoplasmas associated with Flavescence dorée in clematis and grapevine and preliminary results on the role of Dictyophara europaea as a vector

A survey was conducted over several years in Italy and the Balkans in order to gain an understanding of the relationship between the Flavescence dorée (FD) phytoplasma isolates found in clematis and grapevine. A total of 399 clematis and 107 grapevine samples were analyzed. The results showed that 36% of the Clematis vitalba plant samples were infected by phytoplasmas which, in grapevine, are associated with FD, a quarantine disease in Europe. Infected clematis plants were also found in areas where FD phytoplasma had never previously been reported to infect grapevine, such as Macedonia, Croatia and some areas of Italy and Serbia. Molecular data from three phytoplasma genomic fragments showed the presence of different FD phytoplasma isolates, all belonging to the 16SrV-C subgroup, including the Italian FD-C isolate, the isolate found in Serbia, an isolate similar to the French FD2000 and a new isolate typical of central Italy. A few clematis plants were infected with single nucleotide polymorphism, insertion or deletion mutants of the FD-C isolate. Of all the potential Hemipteran vector species surveyed in Italy and Serbia, only 18 of 527 Dictyophara europaea individuals tested proved to be infected with the FD phytoplasma. Preliminary transmission experiments showed that this species is able to transmit the FD phytoplasma from clematis to grapevine. The presence of FD-infected clematis and of D. europaea could, therefore, constitute a risk for FD epidemics in the European viticultural regions.     

桃黄化病植原体昆虫介体的验证及其带毒部位PCR检测

 利用植原体16S rDNA通用引物对采集的北京和天津黄化病桃树总DNA进行巢式PCR检测,证明发病样本的病原为桃黄化病植原体。经过检测昆虫总DNA和经取食过的人工培养液DNA中桃黄化病植原体的16S rDNA,结果表明桃黄化病植原体的有效传播媒介昆虫为桃一点叶蝉。将带毒桃一点叶蝉个体的头部、胸部以及腹部分离,分别在这些部位检测到桃黄化病植原体的16S rDNA,说明桃一点叶蝉的头部、胸部以及腹部都可带毒,表明植原体可从植物汁液进入叶蝉的口针、食道和肠道。     

褐腐病菌三种分子鉴定方法的比较

为了筛选出适用于口岸检疫的、可快速鉴定三种褐腐菌,即美澳型核果褐腐菌(Monilinia fructicola)、核果褐腐菌(M. laxa)和仁果褐腐菌(M. fructigena)的方法,采用Lane等基于形态学特征的方法对采自北京、山东、河北等省市的58株褐腐菌进行了种类鉴定,并以这些菌及三个种的标准菌株为材料比较了已报道的三种分子检测方法的可靠性。研究发现,58株菌中有53株是美澳型核果褐腐菌,2株为核果褐腐菌,3株为仁果褐腐菌。采用Ioos等的PCR方法,从3个标准菌株、53株美澳型核果褐腐菌和2株核果褐腐菌中都只扩增出相应种的特征条带,而从3株仁果褐腐菌中的2株中扩增出了两个种的特征条带。采用Ma等的方法,从美澳型核果褐腐菌标准菌株、采自国内的53株美澳型核果褐腐菌和核果标准菌株中,只扩增到相应种的特征条带,而从采自国内的2株核果褐腐菌株中不仅扩增出了核果褐腐菌的条带还扩增出了美澳型核果褐腐菌的特征条带,3株仁果褐腐菌株产生了核果褐腐菌的特征条带,仁果褐腐菌标准菌株中没有得到产物。采用Cote等的方法,只从16株美澳型核果褐腐菌中扩增出该种的特征条带,从其余菌株(包括标准菌株)中没有获得产物。这些研究结果表明:Ioos等的检测方法可用于检测美澳型核果褐腐菌和核果褐腐菌;Ma等的检测方法可用于检测美澳型核果褐腐菌,而不适用于检测其它两个种;而仁果褐腐菌的分子检测方法需要进一步研究和完善。     

Nested PCR assay for detection of sugarcane grassy shoot phytoplasma in the leafhopper vector Deltocephalus vulgaris: a first report

Nymphs of Deltocephalus vulgaris , the leafhopper vector of sugarcane grassy shoot (SCGS) disease, fed on SCGS-infected and healthy sugarcane leaves, and SCGS-infected and healthy plant tissue of sugarcane cv. CoLk 8102, were examined by nested PCR using phytoplasma-specific rRNA operon primers for detection of the SCGS phytoplasma. Samples of SCGS-infected plants with symptoms and SCGS-exposed D. vulgaris nymphs yielded SCGS-exclusive DNA bands when nested PCR was performed. Negative results were obtained when symptomless plant host and unexposed insect vector samples devoid of phytoplasma DNA templates were used. Such a reliable molecular tool for the precise detection of SCGS phytoplasma in the D. vulgaris population would help forecast the potential of secondary spread of SCGS in a susceptible sugarcane variety, and may facilitate control of the disease.     

温度影响下斑潜蝇种间竞争取代的分子机制研究进展

斑潜蝇是一类为害园艺蔬菜作物的世界性害虫,也是我国重要的外来有害生物。斑潜蝇种类繁多,不断向世界各地入侵扩散,在其扩散过程中种间竞争取代频繁。斑潜蝇种间竞争取代机制复杂,影响种间竞争取代的因子很多,其中温度是影响斑潜蝇种间竞争取代的重要因子。该文首先以温度影响下斑潜蝇种间竞争生态学机制为切入点,分别从关键胁迫耐受性基因——热激蛋白(heat shock protein,Hsp)基因的表达、Hsp基因非编码区的特征和转录组比较分析3个层面对近缘斑潜蝇竞争取代分子机制进行总结与探讨,并从胁迫耐受性基因类型、分子生物学研究方法及种间竞争其他因子等方面对斑潜蝇种间竞争取代未来的研究方向进行了展望。     

Selection and characterization of resistance to Polymyxa betae, vector of Beet necrotic yellow vein virus, derived from wild sea beet

The plasmodiophoromycete Polymyxa betae is an obligate root parasite that transmits Beet necrotic yellow vein virus (BNYVV), the cause of sugar beet rhizomania disease. Currently, control of this disease is achieved through the use of cultivars with monogenic (Rz1) partial resistance to the virus. To improve the level and durability of this resistance, sources of resistance to the virus vector, P. betae, were sought. Over 100 accessions of the wild sea beet (Beta vulgaris ssp. maritima) from European coastal regions were evaluated for resistance in controlled environment tests. Quantification of P. betae biomass in seedling roots was achieved using recombinant antibodies raised to a glutathione‐s‐transferase expressed by the parasite in vivo. Several putative sources of resistance were identified and selected plants from these were hybridized with a male‐sterile sugar beet breeding line possessing partial virus resistance (Rz1). Evaluation of F₁ hybrid populations identified five in which P. betae resistance had been successfully transferred from accessions originating from Mediterranean, Adriatic and Baltic coasts. A resistant individual from one of these populations was backcrossed to the sugar beet parent to produce a BC₁ population segregating for P. betae resistance. This population was also tested for resistance to BNYVV. Amplified fragment length polymorphism and single‐nucleotide polymorphism markers were used to map resistance quantitative trait loci (QTL) to linkage groups representing specific chromosomes. QTL for resistance to both P. betae and BNYVV were co‐localized on chromosome IV in the BC₁ population, indicating resistance to rhizomania conditioned by vector resistance. This resistance QTL (Pb1) was shown in the F₁ population to reduce P. betae levels through interaction with a second QTL (Pb2) found on chromosome IX, a relationship confirmed by general linear model analysis. In the BC₁ population, vector‐derived resistance from wild sea beet combined additively with the Rz1 virus resistance gene from sugar beet to reduce BNYVV levels. With partial virus resistance already deployed in a number of high‐yielding sugar beet cultivars, the simple Pb1/Pb2 two‐gene system represents a valuable additional target for plant breeders.     

Single-strand conformation polymorphism (SSCP), cloning and sequencing reveal a close association between related molecular variants of Grapevine virus A (GVA) and Shiraz disease in South Africa

When a recently discovered 234-nt fragment of the Grapevine virus A (GVA) genome, located in ORF3, was used in single-strand conformation polymorphism (SSCP), it produced patterns of DNA bands which were indicative of GVA variants of molecular group II. This technique of rapid identification of variants of group II was applied to the study of GVA variants in various grapevines with different Shiraz disease (SD) status. The results, supported by nucleotide sequence data, revealed that GVA variants of molecular group II are closely associated with SD in South Africa, and showed that variants of group III are commonly present in SD-susceptible grapevines that consistently do not exhibit symptoms of this disease.     

Development and application of a PCR-based 'molecular tool box' for the identification of Phytophthora species damaging forests and natural ecosystems

A PCR-based 'molecular tool box', based on a region of the ras-related protein gene Ypt 1, was developed for the identification of 15 Phytophthora species that damage forests and trees: P. cactorum , P. cambivora , P. cinnamomi , P. citricola , P. europaea , P. inundata , P. lateralis , P. megasperma , P. nemorosa , P. kernoviae , P. pseudosyringae , P. psychrophila , P. quercina , P. ramorum and P. ilicis . Most primers proved highly specific in blast analyses and in tests with DNA from 72 isolates of 35 species of Phytophthora and nine species representative of Pythium . Exceptions were primers designed for P. cactorum and P. ilicis , which cross-reacted with P. idaei and P. nemorosa , respectively. Amplification with Phytophthora -genus-specific primers before amplification with the various species-specific primers (nested PCR) increased the sensitivity of detection over amplification with species-specific primers only: detection limits ranged between 100 and 10 pg target DNA µ L⁻¹ in the latter, compared with 100 fg µ L⁻¹ in nested PCR. Using existing methods for rapid extraction and purification of DNA, single-round amplification was appropriate for detection of target Phytophthora species in leaves, but nested PCR was required for soil and water samples. The quarantine pathogens P. ramorum and P. kernoviae were detected in a number of naturally infected leaves collected in England and Wales, whereas P. citricola was commonest in water and soil samples from natural Scottish ecosystems.     

Distinguishing sporulating and nonsporulating lesions as a method for evaluating the resistance of sugarcane genotypes to orange rust

Sugarcane breeding programmes rank the resistance of genotypes to Puccinia kuehnii, causal agent of orange rust, according to levels of disease severity. However, during the screening stages, this method of assessment can lead to precipitous elimination of genotypes with promising agronomic traits but showing mild symptoms of rust such as flecks or lesions that do not produce spores. This study aimed to propose a new method to classify the resistance of sugarcane genotypes to orange rust by counting sporulating lesions. Five sugarcane varieties with different levels of resistance to P. kuehnii were inoculated with two pathogen populations under controlled conditions. The disease severity (SEV), total number of lesions (TNL), and total number of sporulating lesions (TNSL) were evaluated in a 20 cm leaf fragment from the most diseased leaf. The TNL and TNSL evaluations were performed at 11, 16 and 21 days after inoculation (DAI) and SEV at 21 DAI. The thresholds of 80% and 8% of sporulating lesions (SL) separated susceptible from the intermediate varieties and intermediate from the resistant ones, respectively. It is proposed that the method of counting sporulating lesions be used in screening genotypes for resistance to P. kuehnii in sugarcane breeding programmes.