Hydrogen peroxide localization and antioxidant status in the recovery of apricot plants from European Stone Fruit Yellows

Hydrogen peroxide (H₂O₂) localization and roles of peroxidases, malondialdehyde and reduced glutathione were compared in leaves of apricot (Prunus armeniaca) plants asymptomatic, European Stone Fruits Yellows (ESFY)-symptomatic and recovered. Nested PCR analysis revealed that Candidatus Phytoplasma prunorum, is present in asymptomatic, symptomatic and recovered apricot trees, confirming previous observations on this species, in which recovery does not seem to be related to the disappearance of phytoplasma from the plant.H₂O₂was detected cytochemically by its reaction with cerium chloride, which produces electron-dense deposits of cerium perhydroxides. H₂O₂was present in the plasmalemma of the phloem cells of recovered apricot plant leaves, but not in the asymptomatic or symptomatic material. Furthermore, by labelling apricot leaf tissues with diaminobenzidine DAB, no differences were found in the localization of peroxidases.Protein content in asymptomatic, symptomatic and recovered leaves was not significantly different from one another. In contrast, guaiacol peroxidase activity had the following trend: symptomatic > recovered > asymptomatic, whereas reduced glutathione content followed the opposite trend: asymptomatic > recovered > symptomatic. Moreover, no differences were observed in malondialdehyde concentrations between asymptomatic, symptomatic and recovered leaves. The overall results suggest that H₂O₂ and related metabolites and enzymes appear to be involved in lessening both pathogen virulence and disease symptom expression in ESFY-infected apricot plants.     

Transmission of Rhus (Rhus javanica L.) Yellows by Hishimonus sellatus and Host Range of the Causal Phytoplasma

In 1998, rhus (Rhus javanica L.) yellows (RhY), caused by phytoplasma, was found in Miyagi Prefecture, Japan. In vector transmission tests, Hishimonus sellatus acquired RhY phytoplasma from diseased R. javanica and transmitted it to healthy R. javanica. Twenty-two species of herbaceous plants in 10 families were infected with RhY phytoplasma by H. sellatus. The host range and main symptoms on test plants of RhY phytoplasma differed from those of Macrosteles striifrons-transmitted phytoplasmas, which belong to the same 16Sr I group phytoplasma. Received 6 December 1999/ Accepted in revised form 14 May 2000     

Detection and Indentification of European Stone Fruit Yellows and Other Phytoplasmas in Wild Plants in the Surroundings of Apricot Chlorotic Leaf Roll-affected Orchards in Southern France

Between 1994 and 1998 a field study was conducted to identify plant hosts of the European stone fruit yellows (ESFY) phytoplasma in two apricot growing regions in southern and southwestern France where the incidence of apricot chlorotic leaf roll was high. A total of 431 samples from 51 different plant species were tested for the presence of phytoplasmas by PCR using universal and ESFY-specific primers. ESFY phytoplasma was detected in six different wild growing Prunus species exhibiting typical ESFY symptoms as well as in symptomless dog rose bushes (Rosa canina), ash trees (Fraxinus excelsior) and a declining hackberry (Celtis australis). The possible role of these plant species in the spread of ESFY phytoplasma is discussed. PCR-RFLP analysis of ribosomal DNA amplified with the universal primers was carried out to characterize the other phytoplasmas found. Thus, elm yellows phytoplasma, alder yellows phytoplasma and rubus stunt phytoplasma were detected in declining European field elm trees (Ulmus carpinifolia Gled), in declining European alder trees (Alnus glutinosa) and in proliferating Rubus spp. respectively. The presence of rubus stunt phytoplasma in great mallow (Malva sylvestris) and dog rose was demonstrated for the first time. Furthermore, the stolbur phytoplasma was detected in proliferating field bindweed (Convolvulus arvensis) and a previously undescribed phytoplasma type was detected in red dogwood (Cornus sanguinea). According to the 16S rDNA-RFLP pattern this new phytoplasma belongs to the stolbur phytoplasmas group.     

Distribution of ‘Candidatus Phytoplasma prunorum’ and its vector Cacopsylla pruni in European fruit‐growing areas: a review

European stone fruit yellows (ESFY) is an EU‐listed I/AII disease affecting Prunus spp. caused by ‘Candidatus Phytoplasma prunorum’. This paper reports the results from a systematic literature review approach that sought to determine the geographic distribution of ‘Ca. Phytoplasma prunorum’ in European fruit‐growing areas. Evidence for the presence of the phytoplasma was found for 15 of the 27 EU countries. It is prevalent in the most important stone fruit production areas of Central and Southern Europe, where it causes substantial impact in apricots (Prunus armeniaca), Japanese plums (P. salicina) and peaches (P. persica). In Northern European areas where these hosts are not produced, it is occasionally found on tolerant species (P. domestica). However, because surveys of the disease status of tolerant hosts are not performed, it remains unclear whether the pathogen is absent in Northern Europe or survives in tolerant cultivated or wild hosts. No reports of ESFY were found from the southernmost part of Europe: Portugal, Spain (Andalucia, Castile–La Mancha), Italy (Sicily, Puglia), Greece (Crete), Cyprus and Malta. This may be explained by the absence of the favoured wild hosts of the vector. Moreover, it remains unclear if the vector finds suitable conditions for aestivation and overwintering in these regions.     

Characteristics of the spread of apple proliferation by its vector Cacopsylla picta

The distribution and natural phytoplasma infection of Cacopsylla picta were investigated during a long-term field survey between 2002 and 2009 in commercial and abandoned apple proliferation-infected orchards throughout Germany, northern Switzerland, and eastern France. Comparable population dynamics were described for the different sites whereas considerable variations in the absolute population densities were observed among the years. Individual polymerase chain reaction (PCR) testing revealed, for each year, a rather stable natural infection rate with ?Candidatus Phytoplasma mali? of ?10% for overwintered adults of C. picta. Both genders were equally highly infected although more females were caught. The overall male/female ratio was 1:1.5. No direct correlation was found between the infection status of the orchard and the infection rate of overwintered C. picta. No influence of agricultural practices was seen. However, a relationship between the incidence of the disease and the vector population density became evident on a regional scale. Successful transmission of ?Ca. P. mali? occurred each year with overwintered individuals as well as with new adults. The transmission efficiency varied among the years within 8 to 45% for overwintered adults and 2 to 20% for individuals of the new generation. The load of single C. picta with ?Ca. P. mali? was determined by quantitative real-time PCR. High phytoplasma titers were measured in overwintered adults already at their first appearance in the orchards after remigration from their overwintering hosts. Thus, the data indicate the transmission of the disease on a regional scale by remigrant adults of C. picta and at a local scale within the same season by emigrant adults which developed on infected plants.     

The insect vector Cacopsylla picta vertically transmits the bacterium ‘Candidatus Phytoplasma mali’ to its progeny

The phloem‐sucking psyllid Cacopsylla picta plays an important role in transmitting the bacterium ‘Candidatus Phytoplasma mali’, the agent associated with apple proliferation disease. The psyllid can ingest ‘Ca. Phytoplasma mali’ from infected apple trees and spread the bacterium by subsequently feeding on uninfected trees. Until now, this has been the most important method of ‘Ca. Phytoplasma mali’ transmission. The aim of this study was to investigate whether infected C. picta are able to transmit ‘Ca. Phytoplasma mali’ directly to their progeny. This method of transmission would allow the bacteria to bypass a time‐consuming reproductive cycle in the host plant. Furthermore, this would cause a high number of infected F₁ individuals in the vector population. To address this question, eggs, nymphs and adults derived from infected overwintering adults of C. picta were reared on non‐infected apple saplings and subsequently tested for the presence of ‘Ca. Phytoplasma mali’. In this study it was shown for the first time that infected C. picta individuals transmit ‘Ca. Phytoplasma mali’ to their eggs, nymphs and F₁ adults, thus providing the basis for a more detailed understanding of ‘Ca. Phytoplasma mali’ transmission by C. picta.     

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.     

Isolation and Characterization of Derivative Lines of the Onion Yellows Phytoplasma that Do Not Cause Stunting or Phloem Hyperplasia

ABSTRACT Two lines of onion yellows phytoplasma producing milder symptoms were isolated from the original line (OY-W). One has an additional characteristic, non-insect-transmissibility (OY-NIM), compared with the other (OY-M). OY-M was established after maintaining OY-W for 11 years on a plant host (Chrysanthemum coronarium) with an insect vector (Macrosteles striifrons), and OY-NIM was isolated after subsequent maintenance of OY-M in plants by periodic grafting. Polymerase chain analysis suggested that OY-NIM cannot traverse the gut or survive in the hemolymph of the leafhopper. OY-W results in witches'-broom formation and stunted growth in the host plant. In contrast, OY-M and OY-NIM do not cause stunting in the host plant, although they result in witches'-broom. Histopathological analysis of these lines revealed that the hyperplastic phloem tissue and severe phloem necrosis seen in OY-W did not exist in OY-M and OY-NIM. This was attributed to a reduction in the population of phytoplasma in tissues in both OY-M- and OY-NIM-infected plants. The results suggest that the cause of stunting and phloem hyperplasia may be genetically different from the cause of witches'-broom. Pulsed field gel electrophoresis analysis showed that OY-M had a smaller genome size ( approximately 870 kbp) than OY-W ( approximately 1,000 kbp). Thus, some of the OY-W genes responsible for pathogenicity may not be present in OY-M.     

Characterisation of a Quantitative Resistance to Vector Transmission of Tomato yellow leaf curl virus in Lycopersicon pimpinellifolium

Two wild genotypes from the same species Lycopersicon pimpinellifolium, WVA106 (susceptible) and INRA-Hirsute (so-called ‘resistant’), were compared with respect to their reaction to Tomato yellow leaf curl virus isolate Réunion (TYLCV-Mld[RE]), using both whitefly-mediated inoculation and graft inoculation. Disease incidence and symptom severity were scored. Presence and quantification of viral DNA were assessed by dot blot hybridisation. Upon insect inoculation, accession INRA-Hirsute showed a moderate resistance against TYLCV that was overcome by a high inoculation pressure obtained by increasing the cumulative number of inoculative whiteflies. Temporal analyses of the disease progress in relation to this criterion exhibited that the protection was quantitative, mainly reducing the TYLCV-Mld[RE] incidence by at maximum 50% at low inoculation pressure. When graft inoculated, the final TYLCV-Mld[RE] disease incidence was 100% in both susceptible and resistant genotypes with severe symptoms, suggesting a reduction of virus transmission by a vector resistance as a possible mechanism. Implications of using such type of resistance in breeding programmes are discussed.     

Latent entry and spread of Colletotrichum acutatum (species complex) in strawberry fields

Anthracnose, caused by Colletotrichum acutatum (species complex), has become a troublesome problem in strawberry production worldwide. This paper reports (i) an optimized sampling method combined with a real‐time PCR technique to detect the latent presence of C. acutatum in cold‐stored strawberry plants used as planting material in several European countries, and (ii) a study of the spread of C. acutatum following a point inoculation under field conditions. Screening of different parts of planting material suggested that C. acutatum is most likely to be present on runners and old petioles. In addition, in seven out of nine batches of planting material from different nurseries, latent infection by C. acutatum was detected in at least one of five replicate samples. Field experiments in 2009 and 2010 showed extensive latent within‐field spread of the pathogen on strawberry leaves, with a within‐row dispersal distance up to at least 1·75 m in 1 week. A straw ground cover between the rows did not decrease C. acutatum spread, probably because introduced (and/or subsequent) inoculum was confined to the plant bed (within the row) and was not present between the beds. Moreover, the number of C. acutatum spores on the symptomless leaves, as estimated using a real‐time PCR method, was significantly (P < 0·05) correlated with the incidence of fruit rot at harvest and post‐harvest (r = 0·56–0·66). These results illustrate the importance of detecting latent infections in planting material and strawberry leaves in the field.     

Incidence and distribution of ‘Candidatus Phytoplasma prunorum’ and its vector Cacopsylla pruni in Spain: an approach to the epidemiology of the disease and the role of wild Prunus

‘Candidatus Phytoplasma prunorum’ is the causal agent of the European stone fruit yellows (ESFY) disease. This phytoplasma affects wild and cultivated species of Prunus to different degrees, depending on their susceptibility. ‘Candidatus Phytoplasma prunorum’ is present in the four regions of Spain surveyed in this study (Aragon, Catalonia, Extremadura and Valencia) with a variable incidence. Results showed that ‘Ca. Phytoplasma prunorum’ was detected in all of the cultivated Prunus species studied, except P. avium and P. dulcis, and was widespread in Spain. The most affected species was P. salicina, with symptoms including early bud break and blooming, leaf curling and yellowing, collapse, and a major decrease in production. In some plots in the Baix Llobregat area of Barcelona province (Catalonia), the incidence of ESFY on P. salicina was as high as 80%. The insect vector, Cacopsylla pruni, was present in all four of the regions studied, with the highest captures in yellow sticky traps in Catalonia on P. mahaleb and in Extremadura in peach orchards. In Baix Llobregat, large populations of C. pruni were present on infected P. mahaleb bushes, and with high infection rates. This was a key factor in the local pathogenic cycle that caused a major ESFY outbreak in the nearby P. salicina orchards. In the Ebro valley (Lleida and Aragon) and Valencia, the surveys showed very low incidences of the disease and low C. pruni populations.     

Transmission of ‘Candidatus Phytoplasma pyri’ by naturally infected Cacopsylla pyri to peach,an approach to the epidemiology of peach yellow leaf roll (PYLR) in Spain

Peach orchards in the northeast of Spain were severely affected in 2012 by a previously unreported disease in this area. The symptoms included early reddening, leaf curling, decline, abnormal fruits, and in some cases death of the peach trees. All the infected peach samples were positive for ‘Candidatus Phytoplasma pyri’, but none were infected by the ‘Ca. Phytoplasma prunorum’. In this work, potential vectors able to transmit ‘Ca. Phytoplasma pyri’ from pear to peach and between peach trees were studied and their infective potential was analysed at different times of the year. Transmission trials of the phytoplasma with potential vectors to an artificial feeding medium for insects and to healthy peach trees were conducted. Additionally, isolated phytoplasmas were genetically characterized to determine which isolates were able to infect peach trees. Results showed that the only insect species captured inside peach plots that was a carrier of the ‘Ca. Phytoplasma pyri’ phytoplasma was Cacopsylla pyri. Other insect species captured and known to be phytoplasma transmitters were present in very low numbers, and were not infected with ‘Ca. Phytoplasma pyri’ phytoplasma. A total of 1928 individuals of C. pyri were captured in the peach orchards, of which around 49% were phytoplasma carriers. All the peach trees exposed to C. pyri in 2014, and 65% in 2015, were infected by ‘Ca. Phytoplasma pyri’ 1 year after exposure, showing that this species is able to transmit the phytoplasma to peach. Molecular characterization showed that some genotypes are preferentially determined in peach.     

A New Natural Planthopper Vector of Stolbur Phytoplasma in the Genus Pentastiridius (Hemiptera: Cixiidae)

A new disease of sugar beet called Syndrome des Basses Richesses, which appeared in Burgundy and Franche-Comté, France, in 1991, is of uncertain aetiology. However, evidence for aerial transmission of the disease, symptom similarity with yellow wilt and preliminary results of phytoplasma detection, support the hypothesis of a phytoplasma being associated to the disease. A search for a natural phytoplasma vector, was conducted in Franche-Comté in 1997 and 1998, in an area where sugar beet crops had been affected since 1996. A cixiid, tentatively identified as Pentastiridius beieri, not described in the preceding years and not formerly reported as a phytoplasma vector, was present in sugar beet plots in high populations from June to August in 1997 and 1998. Individuals were captured and used for transmission experiments to periwinkle and sugar beet seedlings. They were further tested for the presence of a phytoplasma in their body, using PCR amplification of 16S rDNA of phytoplasmas. In 1997 and 1998, from 2% to 13.3% of the individuals carried a stolbur phytoplasma and insects which tested positive, appeared to have transmitted, through feeding, a stolbur phytoplasma to periwinkles and to sugar beets. This cixiid, whose vectoring capacity of stolbur phytoplasma to plants, is now clearly demonstrated, is available for experimental inoculations, in order to examine the role of phytoplasmas in the Syndrome des Basses Richesses, through the observation of symptom expression in phytoplasma-inoculated plants.     

抗根癌菌剂防治核果类果树根癌病的研究

本文利用引进的放射土壤杆菌K84菌株对我国果树根癌病进行了防治效果的研究，主要包括室内、温室和田间抑菌效果的检验、生产用培养基的改进和菌剂剂型的选择等。室内和温室试验表明，K84菌株能够抑制所有供试的生物Ⅱ型和部分生物Ⅰ型的根癌土壤杆菌，而对来自葡萄的Ⅲ型病菌无效。桃树的田间试验表明K84菌株对人工接种的病菌预防效果可达100％，对自然重茬土壤内的根癌菌也有明显的防治效果，苗龄越小、处理越早，防治效果越好。草炭制剂明显优于液体制剂，其保存期长而且易于运输。本文还介绍一种适于大规模生产的培养基WY。     

Rate of Tomato yellow leaf curl virus Translocation in the Circulative Transmission Pathway of its Vector, the Whitefly Bemisia tabaci

ABSTRACT Whiteflies (Bemisia tabaci, biotype B) were able to transmit Tomato yellow leaf curl virus (TYLCV) 8 h after they were caged with infected tomato plants. The spread of TYLCV during this latent period was followed in organs thought to be involved in the translocation of the virus in B. tabaci. After increasing acquisition access periods (AAPs) on infected tomato plants, the stylets, the head, the midgut, a hemolymph sample, and the salivary glands dissected from individual insects were subjected to polymerase chain reaction (PCR) without any treatment; the presence of TYLCV was assessed with virus-specific primers. TYLCV DNA was first detected in the head of B. tabaci after a 10-min AAP. The virus was present in the midgut after 40 min and was first detected in the hemolymph after 90 min. TYLCV was found in the salivary glands 5.5 h after it was first detected in the hemolymph. Subjecting the insect organs to immunocapture-PCR showed that the virus capsid protein was in the insect organs at the same time as the virus genome, suggesting that at least some TYLCV translocates as virions. Although females are more efficient as vectors than males, TYLCV was detected in the salivary glands of males and of females after approximately the same AAP.     

The Effect of Temperature on Variation in Transmission of a BYDV PAV-like isolate by Clones of Rhopalosiphum Padi and Sitobion Avenae

The efficiencies of seven clones of Rhopalosiphum padi and five clones of Sitobion avenae (originating from Greece and the United Kingdom) as vectors of barley yellow dwarf virus (PAV-like isolate) were evaluated at 5, 10 and 15°C. When inoculation took place at 5 or 10°C, clones of R. padi differed in their ability to transmit. At 15°C there were no differences in the vectoring ability of different clones. For S. avenae, there were no interclonal differences in the transmission efficiency at any of the temperatures. The epidemiological consequences of differences in virus transmission at different temperatures are discussed.     

甘蔗白叶病抗病性鉴定方法的建立与应用

为建立简便高效、致病性稳定和规范实用的甘蔗抗白叶病鉴定方法,推动甘蔗抗白叶病育种,本文从甘蔗材料处理与种植、接种液配制、接种方式、病情调查、分级标准制定等层面对甘蔗抗白叶病鉴定技术进行了系统研究与探索,首次优化创建了简便高效、致病性稳定和规范实用的甘蔗抗白叶病鉴定方法,即种苗喷雾接种法和生长期切茎接种法。种苗喷雾接种法：直接筛选甘蔗白叶病植原体蔗茎榨汁加10倍量无菌水配制接种液,接种液喷洒蔗种,塑料薄膜保湿,接种材料桶栽置于防虫温室培养,接种种植30 d开始调查病株率,按1~5级标准进行抗病性评价。切茎接种法：直接筛选甘蔗白叶病植原体蔗茎榨汁加10倍量无菌水配制接种液,鉴定材料桶栽置于防虫温室培养,株龄6月时切茎用移液枪将100 μL接种液滴入根部切口接种,接种种植20 d开始调查病株率,按1~5级标准进行抗病性评价。两种方法与自然传播相似,接种后发病显著、灵敏度高、重现性好,抗性鉴定结果与田间自然发病相吻合。通过两种接种方法和田间自然发病调查,鉴定明确了10个主栽品种对甘蔗白叶病抗病性,其抗病性真实、可靠,可作为今后甘蔗抗白叶病鉴定标准品种。     

欧洲农药市场特点分析

本文分析了欧洲30国的农药销售情况,主要作物上的农药销售情况,以及不同类别农药的销售情况,指出了欧洲农药市场的特点。     

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。