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.     

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.     

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.     

Characteristics and pathogenicity of six Phytophthora isolates from pot plants

From several greenhouse plants showing foot and root rot symptomsPhytophthora isolates were gathered. Isolates fromPeperomia, Saintpaulia andSinningia were identified asP. nicotianae van Breda de Haan var.nicotianae and an isolate fromBegonia asP. cryptogea Pethybr. & Laff. Cardinal temperatures for the various isolates were determined. The specific and non-specific pathogenicity of the isolates was studied by inoculating the different crops with the different isolates, including aP. cryptogea isolate fromGerbera and aP. nicotianae var.nicotianae isolate from carnation. TheP. nicotianae var.nicotianae isolates appeared to be morphologically identical. Some of the isolates were similar in host range, but others exhibited differences in host specificity at 20°C as well as 25, 30 or 35°C. The same applies for the twoP. cryptogea isolates.     

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.     

Detection and partial molecular characterization of Plum pox virus on almond trees in Turkey

Almond (Prunus dulcis) is one of the well known stone fruit species grown for its unripe fruits and delicious seeds in Turkey. In the Trakya region, however, some prevailing virus infections have reduced almond yields and quality. In ten districts of Trakya, 260 leaf samples were collected from affected almond trees in June 2010. DAS–ELISA assays and RT-PCR tests were employed for the identification of viruses. As a result of these detection studies, five of the 260 leaf samples gathered from symptomatic almond trees had Plum pox virus (PPV), 81 of them had Prunus necrotic ringspot virus (PNRSV), and 11 samples contained Prune dwarf virus (PDV). Only four out of 260 samples had a mixture of these viruses. Partial nucleotide sequences of five almond isolates of PPV were determined and compared with 17 other PPV isolates in databases. Computer analysis of obtained and published nucleotide sequences showed identity ranged from 75.72% to 96.87%. Of the five PPV almond isolates obtained, however, there was a close nucleotide identity of 95.82–96.61% to Turkish isolates. Phylogenetic analysis of nucleotides and amino acids showed that five PPV isolates of almond from the Trakya Region of Turkey were clustered in the same subgroup with PPV-T Turkish isolates in GenBank. Therefore we can consider almond isolates of PPV as PPV-T strain, like the two other isolates from apricot trees in Turkey.     

Sensitivity ofPhytophthora Infestans to cymoxanil

The efficacy of cymoxanil, Pulsan (oxadixyl + mancozeb + cymoxanil, 1:7:0.4) and Sandocur-M (oxadixyl + mancozeb + cymoxanil, 1:7:2) in controlling late blight was examined in potatoes (cv. ‘Alpha’). Nine oxadixyl-sensitive and seven oxadixyl-resistant isolates ofPhytophthora infestons from six countries were tested. ED₉₀ values of cymoxanil ranged between 64 and 467 mg a.i./l. No relationship was observed between sensitivity to cymoxanil and sensitivity to oxadixyl. Isolate S6A (1305) from California was the most sensitive to cymoxanil (ED₉₀ = 64 mg/l), whereas isolate R11 from Israel was the least sensitive (ED₉₀ = 467 mg/l). Most isolates from Israel were less sensitive (ED₉₀ = 151–467 mg/l) than isolates from Switzerland, the Netherlands, Ireland, Mexico or California (ED₉₀ = 64–152 mg/l). Three-way mixtures of cymoxanil with mancozeb and oxadixyl were more inhibitory than cymoxanil alone, regardless of the isolate. Sandocur-M was, for most isolates, more effective than Pulsan. The correlation coefficient between sensitivity to cymoxanil and sensitivity to Sandocur-M was 0.67, while that between cymoxanil and Pulsan was 0.76.     

Characterization of Alternaria species associated with leaf blight of sunflower in China

Nine Alternaria species have been reported to be associated with sunflower leaf blight worldwide, and A. helianthi has been recognized as the most prevalent and damaging species. However, the population structure of Alternaria species causing leaf blight of sunflower in China had not been examined thoroughly prior to this study. During 2010 to 2013, a total of 272 Alternaria isolates were obtained from infected sunflower leaves in 11 provinces, autonomous regions, and municipalities of China. Based on morphological traits and sequence analyses of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) and the partial coding sequences of the histone 3 gene, 227 (83.5 %) isolates were identified as belonging to Alternaria tenuissima, the remainder 45 isolates were grouped to A. alternata (16.5 %). Compared with the ITS regions of rDNA, sequence analyses of the partial coding sequences of histone 3 gene displayed a critical role in discrimination of the small-spored Alternaria species. Phylogenetic analysis of the partial coding sequence of histone 3 gene clearly divided the representative Alternaria isolates into two main clades, A. tenuissima and A. alternata. The pathogenicity of A. tenuissima and A. alternata on detached leaves of sunflower cv. Gankui No.2 did not significantly differ between the two species or among isolates from different geographical origins. Our results indicate that the population structure of Alternaria species associated with sunflower leaf blight differed from that reported previously in China since A. helianthi was not found in this study. In addition, this is the first report about A. tenuissima causing leaf blight on sunflower in China.     

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.     

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.     

Wilt and root rot of poinsettia caused by three high-temperature-tolerant Pythium species in ebb-and-flow irrigation systems

Poinsettia plants growing in ebb-and-flow irrigation systems developed wilting and root rot during the summer growing seasons of 2010 in Gifu Prefecture and 2011 in Aichi Prefecture. Pythium species were isolated from roots with rot symptoms. The isolates were identified as P. helicoides and P. myriotylum on the basis of morphological characteristics and sequence homologies in the rDNA internal transcribed spacer regions. In pathogenicity tests, these isolates caused severe wilting and root rot. This is the first report of poinsettia root rot disease caused by P. helicoides and P. myriotylum, although P. aphanidermatum was reported as a pathogen of poinsettia root rot. To better understand these diseases, we performed an epidemiological study of three high-temperature-tolerant Pythium species, P. aphanidermatum, P. helicoides and P. myriotylum. Disease incidence as a percentage of diseased plants was greatest at 35 °C for all three species. Disease severity using the rating scale of root rot was also highest at 35 °C, particularly with high zoospore inoculum densities (100.0 zoospores/mL). Although the disease incidence and severity were reduced at lower temperatures, the three Pythium species were able to cause disease at temperatures as low as 20 °C.     

Effect of avermectin B1 on the development and fecundity of the california red scale,Aonidiella auranth,and on two of its natural enemies:Chilocorus bipustulatus andAphytis holoxanthus

Various immature stages of male and female California red scale,Aonidiella aurantii (Maskell) (Homoptera: Diaspididae), were treated with avermectin B₁ (MK-936) at concentrations ranging from 10 to 100 ppm. Treatments at 10 ppm completely arrested crawler development and 20 and 100 ppm caused 100% mortality of 48-h and 7-day-old male and female 1st instar nymphs, respectively. The development of the 2nd instar was slightly affected by MK-936 at up to 40 ppm. Sublethal doses of the chemical applied to immature stages caused no dramatic reduction in the fecundity of the subsequent, mature females. MK-936 applied to male prepupal and pupal stages and mated females did not prevent male emergence, nor did it affect the ability of males to copulate and inseminate female scales, or the female fecundity. The development and adult emergence of treatedAphytis holoxanthus DeBach (Hymenoptera: Aphelinidae) larvae and pupae parasitizingChrysomphalus aonidum L. scales were not influenced by a 9 ppm MK-936 treatment; leaves sprayed with this concentration had residual toxicity onA. holoxanthus adults for only 24 h post-treatment. Feeding byChilocorus bipustulatus L. (Coleoptera: Coc-cinellidae) on 0.9 ppm MK-936-treated Florida red scales resulted in 100% mortality of 2nd instars and induced sterility in the females.     

Characterization of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melongenae</Emphasis> isolates from Turkey with ISSR markers and DNA sequence analyses

Fusarium oxysporum f. sp. melongenae (Fomg), causal agent of Fusarium wilt of eggplant, is a serious pathogen in open fields and greenhouses. Inter-simple sequence repeat (ISSR) banding profiles, sequence analyses of inter-transcribed-spacer (ITS), translation elongation factor 1-alpha (TEF-1α), and actin (actA) DNA regions were employed in this study to determine genetic diversity and population structure of Fomg isolates obtained from Turkey. For ISSR study, (ACTG)₅, (GACAC)₃, (GACA)₄, (GATA)₄, HVH(TG)₇ and (CA)₈RG primers were selected from a set of 16. Discriminative ability of the primers revealed with various indices including polymorphic information content (PIC), and mean PIC value was calculated as 0.26. The ISSR data revealed 31 loci belonging to 202 Fomg isolates and 14 of them were found to be polymorphic. The isolates on neighbor joining ISSR tree were grouped into two major clusters which separated Fomg and outgroup isolates. Population structure was investigated based on bayesian modeling and results indicated five subpopulations (K = 5, ?K = 205.42). Mean genetic and geographical distances among sampling locations revealed only a weak and insignificant correlation (r = 0.583, P = 0.06). Phylogenetic analyses were carried out with ITS, TEF-1α and actA DNA regions with a selected subset of 30 Fomg, along with one non-host and one outgroup isolates. Since ITS region were not able to provide a meaningful separation, TEF-1α and actA sequences of each organism were concatenated individually to build a dendrogram. The clustering tree successfully separated the Fomg, non-host and outgroup isolates in which all Fomg were located on the same branch, forming a monophyletic group in the dendrogram.     

Response ofSpodoptera littoralis (Boisd.) andEarias insulana (Boisd.) larvae to azadirachtin and salannin

The effect of azadirachtin and salannin, two triterpenoids isolated from seeds of neem (Azadirachta indica A. Juss), on the feeding response ofSpodoptera littoralis (Boisd.) andEarias insulana (Boisd.) larvae, was investigated. Styropor (foamed polystyrene) lamellae were painted on both sides with a mixture of 5% sucrose with different concentrations of either azadirachtin or salannin dissoled in methanol-water (3∶7). Azadirachtin strongly suppressed feeding inS. littoralis larvae even at 0.001%, whereas salannin showed some antifeedant activity at 0.005% and above. Larvae ofE. insulana were deterred from feeding on azadirachtin-treated lamellae even at 0.005%, whereas salannin was effective only at 0.01% and above. Azadirachtin applied on cotton leaves deterred larvae ofS. littoralis from feeding at all concentrations ranging between 0.001 and 0.02%.     

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.     

Genetic diversity of Ralstonia solanacearum infecting solanaceous vegetables from India reveals the existence of unknown or newer sequevars of Phylotype I strains

Bacterial wilt is one of the important constraints in the cultivation of solanaceous vegetables in India. The disease is caused by Ralstonia solanacearum, a soil bacterium. We have collected 232 isolates of R. solanacearum infecting solanaceous vegetables (eggplant, tomato and chilli) and other crops from different parts of India. Pathogenicity of the isolates was tested on eggplant, tomato and chilli and the pathogen was confirmed by PCR. Multiplex PCR and biochemical tests indicated that all the isolates were phylotype I and biovar 3. Ninety-five representative isolates selected based on geographical region, host range and pathogenicity were subjected to further phylogenetic and diversity analysis. Sequence analysis of egl, pga and hrpB genes of 95 isolates and genetic diversity of 50 representative isolates was reported and discussed. Indian isolates within the Phylotype I did not group based on the host or geographical location, except clustering of isolates from the Andaman Islands. Indian isolates clustered into two sub groups based on egl and pga trees indicating the presence of two major population groups. Sub group 1 is the dominant group in the data set and consists of unknown/newer sequevars, and sub group 2 consist of mainly the isolates which are designated with sequevar numbers based on egl sequences. In the hrpB based tree, the sub group 2 is the dominant group in the data set and it is the same for the sub group 1 of the egl tree. Indian phylotpe I R. solanacearum strains are phenotypically diverse including the previously described sequevars 14, 17, 44, 47 and 48. Our studies indicated the existence of R. solanacearum isolates with unknown/newer sequevars; the diversity existing among the phylotype I isolates might be due to a continuous evolutionary process. To our knowledge this is the first detailed report on the diversity of phylotype I R. solanacearum strains infecting solanaceous vegetables and the existence of unknown/newer sequevars in India.     

Comparative toxicity of several insecticides to eggs,larvae and adults of the Egyptian cottonworm,Spodoptera littoralis,in laboratory trials

Insecticides commonly used in cotton fields in Israel against lepidopterous pests were tested against eggs, as well as against 2nd-instar larvae and adults raised from eggs ofSpodoptera littoralis (Boisduval) collected in cotton fields in the Bet She’an Valley. Methomyl, chlorpyrifos, methidathion, monocrotophos, ethyl parathion, and methyl parathion were effective against eggs even at low doses, profenofos and phosfolan were less active, and azinphos-methyl was ineffective. The doses needed for 90% kill (LD ₉₀ ) of the 2nd-instar larvae were 8.5, 35, 280, 1300 and 3400 g a.i./1000 m ² for chlorpyrifos, methomyl, profenofos, methyl parathion and ethyl parathion, respectively; monocrotophos was inactive against 2nd-instar larvae even at relatively high doses. The LD ₉₀ of adults was reached with 16, 32, 1700 and 6100 ga.i./l000 m² of chlorpyrifos, methomyl, ethyl parathion and profenofos, respectively. Only chlorpyrifos and methomyl gave successful control of all three stages of the insect tested, at doses close to those generally used with aerial applications.     

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.