Analysis of genetic and virulence variability of Stemphylium lycopersici associated with leaf spot of vegetable crops

Stemphylium lycopersici (Enjoji) W. Yamam was initially described from tomato and has been reported to infect different hosts worldwide. Sequence analyses of the internal transcribed spacer (ITS) regions 1 and 2, including 5.8S rDNA (ITS-5.8S rDNA) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), as well as virulence studies were conducted to analyze 46?S. lycopersici isolates. Stemphylium lycopersici isolates used in this study were obtained from diseased tomato (Solanum lycopersicum L.), eggplant (Solanum melongena L.), pepper (Capsicum annuum L.) and lettuce (Lactuca sativa L.) from major vegetable growing regions of Malaysia, including the three states of Pahang, Johor and Selangor between 2011 and 2012. Phylogenetic analysis of a combined dataset of the ITS-5.8S rDNA and gpd regions indicated that all isolates were clustered in the sub-cluster that comprised S. lycopersici, and were distinguished from other Stemphylium species. Cluster analyses using the UPGMA method for both RAPD and ISSR markers grouped S. lycopersici isolates into three main clusters with similarity index values of 67 and 68 %. The genetic diversity data confirmed that isolates of S. lycopersici are in concordance to host plants, and not geographical origin of the isolates. All S. lycopersici isolates were pathogenic on their original host plants and showed leaf spot symptoms; however, virulence variability was observed among the isolates. In cross-inoculation assays, the representative isolates were able to cause leaf spot symptoms on eggplant, pepper, lettuce and tomato, but not on cabbage.     

Arthropod fauna and main insect pests of plane trees in Israel

Fifty-four insect and four mite species are included in a list of the entomofauna of plane trees in Israel. Only two species are monophagous:Phyllonorycter platani (Stgr.) (Lepidoptera: Gracillariidae) andEdwardsiana iranicola Zachv. (Heteroptera: Cicadellidae). Four species are noxious:P. platani, the main insect pest of the plane trees in Israel;Zeuzera pyrina L. (Lepidoptera: Cossidae);Kalotermes flavicollis F. (Isoptera: Kalotermitidae); andE. iranicola. Of much lesser importance areTargionia vitis Sign. (Homoptera: Diaspididae),Heliothrips haemorrhoidalis Bché. andRetithrips syriacus May. (Thysanoptera: Thripidae). About half of the listed species are natural enemies and many are parasites ofP. platani. Details are given on the noxious species, together with recommendations for prevention and control.     

Development of conventional and real-time PCR protocols for specific and sensitive detection of Colletotrichum capsici in chilli (Capsicum annuum L.)

Anthracnose, caused by Colletotrichum capsici, is a major disease of chilli (Capsicum annuum L.) affecting both fruit and seed quality. The pathogen is both internally and externally seedborne. However, a rapid and sensitive method for detection of this pathogen in seeds is currently limited. In this study, a polymerase chain reaction (PCR) method based on sequence characterized amplified region (SCAR) marker was developed for specific and sensitive detection of C. capsici in chilli seeds and fruits. The developed SCAR primers were highly specific to C. capsici and resulted in the amplification of an expected 250-bp fragment from genomic DNA of all seven of the C. capsici isolates tested. No amplification occurred when the SCAR primers were tested with genomic DNA from three other fungal isolates and four other Colletotrichum species. The SCAR primers successfully amplified similar sized fragments from DNA derived from C. capsici-infected chilli fruits. The molecular detection sensitivity of C. capsici was 1 pg of purified C. capsici DNA template and 25 ng of DNA from C. capsici-infected chilli fruits. A real-time PCR assay was also developed using SYBR Green chemistry for detection of C. capsici in chilli fruits and seeds. The standard curve obtained showed a linear correlation between copy number of the cloned target DNA sequence of C. capsici and cycle threshold (Ct) values, with R² of 0.98. These PCR-based assays may be highly useful in detection of this important pathogen in chilli seeds and fruits in plant quarantine laboratories.     

Phenotypic and genotypic structure of Phytophthora infestans populations on tomato and potato in the North of Thailand in 2000–2002

A total of 80 single–lesion isolates of Phytophthora infestans were collected from tomatoes and potatoes in several locations in Chiang Mai and Tak provinces in 2000–2002. These isolates were analyzed for mating type, metalaxyl sensitivity, mitochondrial DNA (mtDNA) haplotype RFLP pattern as determined by probe RG57, and for microsatellite markers. All isolates were A1 mating type. Isolates from tomato were usually sensitive to metalaxyl, but isolates from potato were usually resistant to metalaxyl. With one exception, all tomato isolates were related to the US-1 clonal lineage. With two exceptions, all potato isolates were related to two European lineages. In these two provinces, the populations of P. infestans on tomatoes are clearly different from those on potatoes.     

Plant ferredoxin-like protein enhances resistance to bacterial soft rot disease through PAMP-triggered immunity in Arabidopsis thaliana

The protection of plants against bacterial disease is one of the important issues that need to be studied in agricultural applications. The application of a transgene, such as a gene that encodes plant ferredoxin-like protein (PFLP), to generate resistant plants is one possible strategy. Our previous reports have demonstrated that transgenic plants that express extracellular PFLP (ESF plants) are more resistant to bacterial pathogens. This protein intensifies the hypersensitive response (HR) in plants when they are infiltrated by a pathogen-associated molecular pattern (PAMP), harpin (HrpZ), from Pseudomonas syringae. In addition, this intensified HR is associated with the expression of membrane-bound NADPH oxidase. Thus, we attempted to determine the involvement of PFLP in intensifying PAMP-triggered immunity (PTI) to enhance disease resistance. First, we showed that transgenic Arabidopsis plants with the pflp gene were resistant to bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc). Then, the fliC gene which encoded flagellin from Pcc was cloned and expressed. The FliC protein was used in the functional study with PFLP in Arabidopsis Col-0 plants. The reactive oxygen species (ROS) generation and HR ratio were induced by the treatment with both PFLP and FliC together, but they were not induced by treatment with PFLP or FliC alone. Similar results were confirmed in ESF plants, where FliC elicited rapid ROS accumulation and callose deposition. Moreover, we demonstrated that the PFLP-intensified ROS generation and HR were related to Ca²⁺ influx and activation of NADPH oxidase. We concluded that the PFLP-intensified disease resistance is associated with the intensification of PAMP-triggered immunity.     

First record of the red gum lerp psyllid,Glycaspis brimblecombei Moore (Hemiptera Psyllidae), in Tunisia

During a survey carried out in August 2013 along all coastal areas of north-eastern Tunisia (governorships of Bizerte, Ariana, Tunis, Ben Arous, Nabeul, Sousse), eucalyptus trees were found to be highly infested by the invasive pest Glycaspis brimblecombei Moore, 1964, also known as red gum lerp psyllid. This insect, native to the Australian region and secondarily dispersed also in the Americas, Mauritius, Madagascar and South Africa, very recently started to invade the Mediterranean region and in almost 5 years has spread to the Iberian Peninsula, Italy, Greece and Morocco. Its presence in Tunisia (which is recorded here for the first time) most probably dates back to summer 2012, since typical necrotic spots caused by the lerp of the psyllid had already been noted on leaves during spring 2013. No presence of its main parasitoid – Psyllaephagus bliteus Riek – nor of any other natural enemy, was noted up to now during our survey in Tunisia.     

A SCAR marker specific for rapid detection of the avirulence gene Avr1c in Phytophthora sojae

The F₂ population derived from a cross between isolates pR_x (Avr1c-Avr1c) and ps₁ (avr1c-avr1c) of Phytophthora sojae, fungal agent of soybean stem and root rot, was used to determine the genetic basis of avirulence towards Rps1c gene in soybean. The results indicated that this avirulence is dominant and controlled by a single locus, as expected for a simple gene-for-gene model. Segregation of Avr1c in the F₂ progeny of this cross fits a 3:1 ratio. Four of 80 AFLP primers effectively distinguished the avirulent pR_x from the virulent ps₁. Among the 5 specific markers, band C was amplified from the avirulent pR_x by primer set EGC/MAT, then recovered and cloned. This AFLP marker was successfully transfered to a SCAR marker through sequencing, primer design and specific amplication of the DNA of the avirulent pR_x. Results of validity and specificity experiments with 50 individuals of the F₂ progeny and 50 field isolates demonstrated that this SCAR marker (a 616-bp fragment) can be successfully and specifically amplified from the P. sojae isolates that have Avr1c gene.     

Survey on viroids infecting grapevine in Italy: identification and characterization of Australian grapevine viroid and Grapevine yellow speckle viroid 2

Five viroid species have been reported from grapevine. Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd-1) are distributed worldwide, whereas Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd) and Citrus exocortis viroid (CEVd) are found only sporadically. However, the presence of AGVd and GYSVd-2 in several countries, including China, Turkey and Tunisia, suggests a wider dissemination, possibly also in Europe, where AGVd has never been found and GYSVd-2 has been occasionally identified in Italy. Taking advantage of a multiplex RT-PCR assay recently developed for detecting simultaneously these five viroids, vines growing in Italy in commercial vineyards and germplasm collections were surveyed. Besides confirming the widespread presence of HSVd and GYSVd-1 in the field, GYSVd-2 and/or AGVd were identified in two grapevine table cultivars (Sultanina Bianca and Red Globe) from germplasm collections. Tests extended to vines cultivated in southern Italy confirmed the presence of both viroids, which were further characterized. No major sequence divergences between the AGVd and GYSVd-2 variants from Italy and those previously described from other countries were observed. Phylogenetic analysis supported the close relationships among AGVd variants from Italy, Tunisia and Australia. To our knowledge this is the first report of AGVd in Europe and the first molecular characterization of GYSVd-2 isolates from a European country.     

Identification and species status of the mango biotype of Colletotrichum gloeosporioides in Ghana

Research work was carried out to identify and ascertain the species status of the mango biotype of Colletotrichum gloeosporioides infecting mangoes in Ghana. Forty five isolates of Colletotrichum species were collected from 12 districts in Ghana while five each were obtained from mango fruits from Florida, Mexico and Puerto Rico. The entire internal transcribed spacer region, partial beta-tubulin gene and partial glyceraldehyde-3-phosphate dehydrogenase gene of isolates were sequenced and used in phylogenetic studies. The results of the sequence analysis of the first ribosomal transcribed spacer (ITS 1) region showed that 35 % of the isolates from Ghana and all the five isolates from Mexico were the mango biotype of C. gloeosporioides, while the others were not. Phylogenetic studies showed that the mango biotype of the pathogen was Colletotrichum asianum but not C. gloeosporioides as previously thought. However, the other isolates that were not the mango biotype were identified as Colletotrichum siamense and Colletotrichum species which had probably cross-infected mango from other fruit crops in the field.     

Molecular variability of Apple chlorotic leaf spot virus in Shaanxi,China

Apple chlorotic leaf spot virus (ACLSV) is one of the latent viruses that occur in apple orchards worldwide but usually without visible symptoms. In 2010–2012, a total of 550 apple leaf samples from 12 different major apple-producing areas in Shaanxi, China, were tested by serological assay for ACLSV; the results revealed an infection level of 51.5 %. Because of the known variability in the putative amino acid sequences of the coat protein (CP), and thus the potential for non-detection by serological assay, the molecular variability of isolates of ACLSV collected in Shaanxi was analyzed using PCR and compared with isolates from the rest of the world. Sequences of 504 nt corresponding to 87 % of the CP gene of 12 isolates were acquired by RT-PCR and deposited in GenBank with the accession numbers KF134387–KF134298. Comparisons of the partial CP gene sequences of these 12 isolates as well as isolates previously reported in the world revealed the pairwise identities ranging from 68.9–99.8 % and 73.8–100 % at the nucleotide and amino acid level, respectively. Phylogenetic analysis based on these nucleotide sequences showed that the 72 isolates deposited in GenBank fell into three groups (P205, B6 and Ta Tao 5 Group). Our 12 ACLSV isolates were separated into the P205 and B6 groups, respectively. Multiple alignment analysis of the amino acid sequences of CP revealed that there was a combination of six amino acids at positions 40, 59, 75, 86, 130 and 184 in isolates from each group that could be used to distinguish among the three groups. Two recombination events were identified from all isolates by recombination analysis, and three ACLSV isolates collected in this study participated in these two events. Our results show that molecular variation was present in isolates of ACLSV collected in Shaanxi province and this may reflect introductions of the virus associated with different sources of germplasm.     

Braconidae (Hymenoptera) associated with forest and ornamental trees and shrubs in Israel

Data are presented on the occurrence of Braconidae (Hymenoptera parasitica) parasitizing insects associated with forest and ornamental trees and shrubs in Israel. Fifty-five genera of plants are listed, the richest in braconid fauna being tamarisk (9 species); acacia, pistachio and poplar (8 species each); carob and oak (7 species each). Of the 95 species of insect hosts mentioned, 53 are Lepidoptera, mostly Gelechiidae (7 species), Pyralidae (6 species), Noctuidae (5 species), Gracillariidae, Tortricidae, Geometridae, Lymantriidae and Lycaenidae (3 species each); 44 are Coleoptera, mostly Cerambycidae (13 species), Scolytidae (12 species), and Bostrichidae (9 species); three are Diptera. Of the 92 species of braconids listed, of which only 65 have been fully named, 56 develop in Lepidoptera, mostly Noctuidae (15 species), Gelechiidae (11 species) and Pyralidae (9 species); 33 species develop in Coleoptera, mostly Cerambycidae (12 species, Bostrichidae (10 species) and Scolytidae (5 species); and three species develop in Diptera. Thirty-eight species are new to the fauna of Israel; at least three of them are new to science,viz., Gnaptodon, Gildoria andDendrosotinus titubatus Papp.     

Detection and estimation of Citrus Tristeza Virus infection rates based on Elisa Assays of packing house fruit samples

Enzyme-linked immunosorbent assay (ELISA) proved to be a sensitive detector for citrus tristeza virus (CTV) in orange fruits (Citrus sinensis (L.) Osbeck). Samples of five fruits were taken from 350-kg packing house containers and tested by ELISA to predict the infection rate of CTV in two infected orange groves. The predicted infection rates, 1% and 11%, were in reasonable agreement with the observed rates of 1% (15/1400) and 16% (324/2053), respectively. The 360 test samples from reputedly uninfected groves all tested negative. These results suggest that the ELISA procedure may provide a general method of detecting viral or other systemic pathogenic infections using the fruit as the test material in place of tree tissue. Fruit samples can be collected routinely at the packing house to reduce test costs.     

Alien scale insects (Hemiptera Coccoidea) in European and Mediterranean countries: the fate of new and old introductions

This contribution focuses on recent interceptions and introductions of alien scale insects and their current distribution in European and Mediterranean countries. Data and collections were gathered in markets, nurseries, and botanical gardens, mostly in Italy, either indoors or outdoors. New or recent records of the following alien species are presented: Exallomochlus hispidus (Morrison); Ferrisia virgata (Cockerell) (Pseudococcidae); Coccus viridis (Green); Milviscutulus mangiferae (Green) (Coccidae); Aonidiella orientalis (Newstead); Aspidiotus destructor Signoret; Aulacaspis tubercularis Newstead; Fiorinia fioriniae Targioni Tozzetti; Lepidosaphes pinnaeformis (Bouché); Pseudaulacaspis brimblecombei Williams (Diaspididae). New data and pest status of Phoenicococcus marlatti Cockerell (Phoenicococcidae) and Trabutina mannipara (Hemprich & Ehrenberg) (Pseudococcidae) are also reported. The possible repeated introductions of the latter from North Africa to south Italy by trans-Mediterranean winds, is hypothesized.     

Control of phytophthora leaf blight of taro (Colocasia esculenta) by fungicides and roguing

Six fungicides were evaluated under laboratory and field conditions for control of Phytophthora leaf blight of taro,Coloeasia esculenta, incited byPhytophthora colocasiae. Inin vitro tests Deraosan 65W was the most effective in inhibiting the mycelial growth of the test pathogen followed by Difolatan 80W, Fytolan (copper oxychloride), Apron 35F, Topsin-M 50W and Dithane Z-78 75W. Excellent control was obtained with Demosan 65W and Difolatan 80W, good control with Apron 35F, fair control with Fytolan, and poor control with Topsin-M 50W and Dithane Z-78 75W. Results ofin vivo tests were correlated with those of thein vitro tests. Roguing of infected leaves did not eradicate the pathogen but can only delay epiphytotics.     

Serological recognition of Streptomyces species causing scab on potato tubers

Antisera were prepared against extracts of two tyrosinase-positiveStreptomyces spp., one of which caused a “deep” and the other a “russet” scab. Tyrosinase-positiveStreptomyces isolates not reacting with either of these antisera proved to be nonpathogenic to potato tubers, with few exceptions only. Not all isolates reacting with one or both antisera, however, were pathogenic and so all the serological positive ones had to be tested for pathogenicity to potato tubers. To obtain this relative specificity the antisera had to be absorbed with an extract of a non-pathogenic tyrosinase-positive isolate.     

Biological and Serological Characterization of Zucchini Yellow Mosaic and Watermelon Mosaic Virus-2 Isolates in Israel

Cucurbit potyviruses were collected in the field in Israel and subcultured in indicator plants in a greenhouse. Partial characterization of the Israeli cucurbit potyviruses was done on the basis of host reaction using cucurbits, peas andChenopodium spp. as hosts. Further classification of potyviruses was done by enzyme-linked immunosorbent assay (ELISA) and serological specific electron microscopy (SSEM). By these methods it was possible to identify three of the four isolates as strains of the zucchini yellow mosaic virus, while the fourth was identified as watermelon mosaic virus-2. Two of the ZYMV isolates were nonaphid-transmissible following prolonged mechanical transmission in a greenhouse. Both of these isolates were found to produce helper components capable of assisting the transmission of virions from a transmissible isolate but not those of their own.     

The overwintering mode ofBemisia tabaci and its parasitoids in Israel

Wild and cultivated plants in the vicinity of Kibbutz Nahshon and a few additional locations in Israel were sampled for the presence ofBemisia tabaci Genna-dius (Homoptera: Aleyrodidae). The whiteflies, together with their parasites,Eretmocerus mundus andEncarsia lutea, were found to develop on numerous host species throughout the winter. Especially high levels were reached onLan-tana camara, Abutilon grandifolium andIpomoea batatas. During late winter and spring the population on these hosts declined. From April onwards the populations increased on potatoes and sunflowers.     

Evaluation of biochemical methods for the diagnosis of the avocado sunblotch viroid in Israel

The reliability of biochemical diagnostic methods for avocado sunblotch viroid (ASBV) was evaluated for the Israeli avocado propagation program. Polyacry-lamide gel electrophoresis (PAGE) was compared with hybridization toin vitro ³²P-labeled cDNA and ASBV-RNA probes. Although hybridization to a cDNA probe was the most sensitive method, not all known infected plants were detected. In the light of these results, the problem of diagnosing ASBV in the Israeli propagation program is discussed.     

Sensitive detection of two plant viruses by enzyme-amplified elisa

Enzyme-amplified ELISA (enzyme-linked immunosorbent assay) increased the sensitivity of detection of citrus tristeza virus and papaya ringspot virus in plant sap by 25- and 125-fold, respectively, compared with direct double antibody sandwich ELISA. The advantages of the new substrate system for the detection of low concentrations of viruses in plants are discussed.