A comparison of inoculation techniques for inducing aflatoxin contamination and<Emphasis Type="Italic">Aspergillus flavus</Emphasis> kernel infection on corn hybrids in the field

Aspergillus flavus inoculation techniques were compared on aflatoxin-resistant and -susceptible corn hybrids for inducing aflatoxin contamination andA. flavus kernel infection. A dry carrier technique was comparable to the standard inoculation techniques (the side-needle and a spray technique) in differentiating between the resistant and the susceptible hybrids in the first year of the study. However, only hybrids inoculated with the side-needle technique had statistically different levels of aflatoxin andA. flavus kernel infection in the second year of the study. In a second study, a modified pinbar technique with inoculations near the tip or base of the ear was compared with the side-needle technique. When developing ears were inoculated near the base with the modified pinbar, adequate levels of aflatoxin were induced both years to distinguish between the resistant and susceptible corn hybrids. The modified pinbar technique has the potential of being a useful tool in evaluating corn germplasm for aflatoxin resistance. http://www.phytoparasitica.org posting May 4, 2007.     

Is the parasitization rate of<Emphasis Type="Italic">Diaeretiella rapae</Emphasis> influenced when<Emphasis Type="Italic">Brevicoryne brassicae</Emphasis> feeds on<Emphasis Type="Italic">brassica</Emphasis> plants?

The development time and parasitization rate ofDiaeretiella rapae (M’Intosh) onBrevicoryne brassicae (L.) feeding on differentBrassica cultivars was studied in the laboratory at 20°C. The shortest development time from egg to adult parasitoid was 11.6 days on cabbage cv. ‘Yalova 1’ and the longest was 12.1 days on turnip cv. ‘Antep’ and rapeseed cv. local variety. Females lived significantly longer than males on the host plants used in the study. Females and males had the shortest longevity on rapeseed at 11.1 and 5.1 days, respectively. The highest percent parasitism ofB. brassicae byD. rapae was found on cabbage (40.20%), and the lowest was recorded on turnip (32.64%). Our results demonstrate that parasitism rate could be influenced by the plant quality, probably due to the nutritional status of the aphids or to toxic compounds ingested through the plant. Cabbage, cauliflower and broccoli were found to be suitable plants for the parasitoid, considering the development time of pre-adults, and the parasitization rate ofD. rapae onB. brassicae. http://www.phytoparasitica.org posting Jan. 23, 2007.     

Analysis of protease activity in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">A. parasiticus</Emphasis> on peanut seed infection and aflatoxin contamination

Aspergillus flavus and A. parasiticus are aflatoxin-producing fungi that can infect peanut seeds in field crops. An association between A. parasiticus proteolytic enzyme activities and peanut fungal infection was examined. For this study, a model of inductive and non-inductive culture media to produce A. parasiticus extracellular protease before infection was used. These A. parasiticus cultures were used to infect peanut seeds of cultivars resistant and susceptible to aflatoxin contamination. Peanut seeds of both cultivars exposed to fungi grown on casein medium (inductive medium) showed higher internal and external infection and a higher fungal protease content than those observed on potato dextrose agar (PDA) and sucrose medium (non-inductive media). A further study showed higher fungal colonisation and aflatoxin contamination in seeds of the resistant cultivar pre-incubated with Aspergillus extracellular proteases than in those incubated without proteases. Moreover, protease activities affected the viability of non-infected resistant cultivar seeds, inhibiting germination and radicle elongation and enhancing seed tissue injury. The results strongly suggest that protease production by A. parasiticus is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa. The analysis of fungal extracellular proteases formed on peanut seed during infection showed that A. flavus and A. parasiticus produced metallo and serine proteases; however, there were differences in the molecular masses of the enzymes between both species. The greatest activity in both species was by serine protease, that could be classified as subtilase.     

Effect of seed treatment of<Emphasis Type="Italic">Arachis hypogaea</Emphasis> with<Emphasis Type="Italic">Bacillus subtilis</Emphasis> on nodulation in biocontrol of southern blight (<Emphasis Type="Italic">Sclerotium rolfsii</Emphasis>) disease

The employment of formulatedBacillus subtilis for peanut seeds (Arachis hypogaea cv. ‘Shulamit’) counteracted the destructive effects of the seedborne pathogenSclerotium (Athelia)rolfsii on the nodulation, leghemoglobin and nitrogenase activity of peanuts. Moreover, the changes in crop vigor index, total nitrogen content and survivability of bothRhizobium spp. andB. subtilis have been related to compatibility and even an occasional synergism between them. http://www.phytoparasitica.org posting Nov. 12, 2006.     

Behavior and mutation of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in <Emphasis Type="Italic">Solanum toxicarium</Emphasis> grown in aseptic culture

To study the behavior and mutation of Ralstonia solanacearum in Solanum toxicarium, which is resistant to bacterial wilt, S. toxicarium was grown in aseptic culture and inoculated with R. solanacearum. Although 60%–80% of the inoculated plants were wilting after 2 to 3 days, most wilted plants had recovered by 20 days after inoculation. The pathogen was reisolated from over 98% of inoculated plant stems, but the percentage of recovery decreased the closer the isolation sites were toward the upper stem sections. Three colony types, characterized as fluidal white, nonfluidal red, and a mixture of fluidal white and nonfluidal red, were reisolated from the stems. Nonfluidal red colonies were less virulent on tomato plants than fluidal white colonies.     

<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> and <Emphasis Type="Italic">P. peruviana</Emphasis>, new natural hosts of <Emphasis Type="Italic">Tomato chlorosis virus</Emphasis>

Tomato chlorosis virus causes yellow leaf disorder epidemics in many countries worldwide. Plants of Physalis ixocarpa showing abnormal interveinal yellowing and plants of Physalis peruviana showing mild yellowing collected in the vicinity of tomato crops in Portugal were found naturally infected with ToCV. Physalis ixocarpa and P. peruviana were tested for susceptibility to ToCV by inoculation with Bemisia tabaci, Q biotype. Results confirmed that ToCV is readily transmissible to both species. The infection was expressed in P. ixocarpa by conspicuous interveinal yellow areas on leaves that developed into red or brown necrotic flecks, while P. peruviana test plants remained asymptomatic. Infected plants of both P. ixocarpa and P. peruviana served as ToCV sources for tomato infection via B. tabaci transmission. This is the first report of P. ixocarpa and P. peruviana as natural hosts of ToCV.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

Note: Does<Emphasis Type="Italic">Liriomyza cicerina</Emphasis> affect the yield of chickpeas (<Emphasis Type="Italic">Cicer arietinum</Emphasis>)?

Liriomyza cicerina (Rondani, 1875) (Diptera: Agromyzidae) is an important pest on chickpea (Cicer arietinum L.) in some regions of Turkey. The objective of this study was to determine whether the populations ofL. cicerina on different varieties of chickpea plants during the 2004 and 2005 production seasons affected yield in Sanliurfa province. The trials were carried out using eight different varieties of chickpea with three replicates. During each season, larval densities on leaves were assessed weekly. TheL. cicerina larval population was lowest on four varieties for both seasons. There were very minor differences in yield among the eight varieties in the production seasons. There was no correlation found between larval density and yield loss. http://www.phytoparasitica.org posting Jan. 14, 2007.     

Environmental persistence of<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> products tested under natural conditions against<Emphasis Type="Italic">Thaumetopoea wilkinsoni</Emphasis>

Information on persistence ofBacillus thuringiensis (Bt) is needed to improve the microbial pest management programs against the pine processionary mothThaumetopoea wilkinsoni in pine forests in Israel. The persistence of the microbe under natural conditions of rain and sunlight was evaluated and is documented here for the first time. Pine saplings were sprayed with three commercialBt products, Foray 48B, Delfin WG and Dipel DF, all used at 32,000 IU mg⁻¹ in a formulation with 1% (w/v) of condensed milk. In experiments conducted in November and December of 2004, the saplings were either exposed to rain and sunlight or were sheltered to avoid these environmental factors. The lowest rainfall recorded in the 8-day experiments was 16.5 mm (test 2) and the heaviest was 71.1 mm (test 1). Solar irradiation ranged from 9.4 to 10.9 MJ m⁻². The minimum temperature was close to 10°C and the maximum was less than 23°C. Needles of the treated saplings and their controls were sampled after 0, 1, 5 and 8 days, and were fed to 1 ^st or 2 ^nd instar larvae. Dipel DF persisted better than Delfin WG and still retained its initial activity of 80–100% mortality on day 8 at low rainfall (test 2). Dripping ofBt from upper to lower branches was quantified with the larval bioassays. The milk formulation proved to be an effective rain-fasting adjuvant. http://www.phytoparasitica.org posting May 4, 2007.     

Differing susceptibility to<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> formulations of<Emphasis Type="Italic">Thaumetopoea wilkinsoni</Emphasis> populations between forests with different<Emphasis Type="Italic">Bt</Emphasis> management in Israel

The susceptibility ofThaumetopoea wilkinsoni larvae toBacillus thuringiensis (Bt) formulations was screened in 2003 and 2004. Eggs and larvae were collected from pine forests in 11 geographical locations in Israel. Larval mortality bioassays were conducted with commercial formulations (Delfin WG, Dipel DF and Foray 48B) at concentrations ranging from 0.001% to 0.1%. Significant differences in susceptibility toBt were recorded among populations that were treated withBt intensively, frequently, or never. The mortality recorded in a population that was never treated withBt was twice that in an intensivelyBt-treated population. The correlation between susceptibility toBt and the possible resistance to the microbe is discussed. http://www.phytoparasitica.org posting Jan. 31, 2007.     

Note: Pyrethroid resistance and possible involvement of glutathione<Emphasis Type="Italic">S</Emphasis>-transferases in<Emphasis Type="Italic">Helicoverpa armigera</Emphasis> from Turkey

An introductory study was conducted to investigate the pyrethroid resistance ofHelicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) strains in Turkey, collected from cotton fields in the Adana and Antalya provinces, through two different synthetic pyrethroid insecticides: lambda-cyhalothrin and esfenvalerate. In addition, the roles of glutathioneS-transferases (GSTs) in this resistance mechanism were analyzed. It was found that whereas resistance ratios for lambda-cyhalothrin (LD₅₀ levels) were 3- and 98-fold increased in the Adana and Antalya strains, respectively, esfenvalerate ratios were 3.3- and 92.3-fold increased in the Adana and Antalya strains, respectively, with respect to the susceptible strain. Furthermore, Adana and Antalya strains showed 2.4- and 2.9-fold higher GST activities than the susceptible strain, respectively. In the Antalya field strain, the minor increase in GST activity compared with the resistance levels implies that GSTs may be not greatly involved in this resistance. It also provides evidence that they could not be the only metabolic mechanism responsible for resistance to lambda-cyhalothrin and esfenvalerate inH. armigera from Turkey. http://www.phytoparasitica.org posting Nov. 16, 2006.     

Host status and reaction of <Emphasis Type="Italic">Medicago truncatula</Emphasis> accessions to infection by three major pathogens of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>)

Ditylenchus dipsaci, the stem nematode of alfalfa (Medicago sativa), Mycosphaerella pinodes, cause of Ascochyta blight in pea (Pisum sativum) and Aphanomyces euteiches, cause of pea root rot, result in major yield losses in French alfalfa and pea crops. These diseases are difficult to control and the partial resistances currently available are not effective enough. Medicago truncatula, the barrel medic, is the legume model for genetic studies, which should lead to the identification and characterization of new resistance genes for pathogens. We evaluated a collection of 34 accessions of M. truncatula and nine accessions from three other species (two from M. italica, six from M. littoralis and one from M. polymorpha) for resistance to these three major diseases. We developed screening tests, including standard host references, for each pathogen. Most of the accessions tested were resistant to D. dipsaci, with only three accessions classified as susceptible. A very high level of resistance to M. pinodes was observed among the accessions, none of which was susceptible to this pathogen. Conversely, a high level of variation, from resistant to susceptible accessions, was identified in response to infection by A. euteiches.     

Analysis of sources of oxolinic acid-resistant field strains of <Emphasis Type="Italic">Burkholderia glumae</Emphasis> based on rep-PCR analysis and nucleotide sequences of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Repetitive sequence-based polymerase chain reaction (rep-PCR) analysis using BOX and ERIC as primers showed a highly divergent phylogeny among field strains of Burkholderia glumae. To elucidate the sources of oxolinic acid (OA) resistance in field strains of B. glumae isolated from rice seedlings cultivated in Mie, Toyama, and Iwate prefectures, Japan, the amino acid at position 83 of GyrA (GyrA83), which is involved in OA resistance, and the DNA patterns from the rep-PCR and the partial nucleotide sequences of gyrB and rpoD from various strains were analyzed. The ten Mie strains, in which GyrA83 was isoleucine (Ile), were divided into two groups based on the band patterns in rep-PCR analysis, although the nucleotide sequences of gyrB and rpoD were identical among the strains. Based on the band patterns in the rep-PCR analysis and the gyrB and rpoD sequences, two highly OA-resistant Toyama strains, Pg-13 and Pg-14, for which GyrA83 was serine (Ser) and Ile, respectively, were in the same lineage. This suggests that the bacteria might acquire OA resistance faster than phylogenic diversity as determined with the repetitive sequences BOX and ERIC and with gyrB and rpoD. Furthermore, three Iwate strains (H95, H101, and H104), isolated from seedlings of different cultivars grown in different years and having Ile at GyrA83, are probably in the same lineage, suggesting that OA-resistant bacteria might be transferred among different cultivars.     

Susceptibility and resistance of <Emphasis Type="Italic">Beta vulgaris</Emphasis> subsp. <Emphasis Type="Italic">maritima</Emphasis> to foliar rub-inoculation with <Emphasis Type="Italic">Beet necrotic yellow vein virus</Emphasis>

Four lines (designated MR0, MR1, MR2, and M8) from 13 accessions of Beta vulgaris subsp. maritima were selected on the basis of phenotypes produced after foliar rub-inoculation with Beet necrotic yellow vein virus (BNYVV). The susceptible phenotype developed bright yellow local lesions, whereas the resistant phenotype had symptoms ranging from no visible lesions to necrotic lesions at the inoculation site. MR1 and MR2 lines had a resistant phenotype depending on the isolate and the MR0 line was susceptible to all isolates of BNYVV tested. The M8 line was highly susceptible; the virus spread systemically and caused severe stunting. These plant lines will be useful for distinguishing BNYVV isolates having different pathogenicities, especially those controlled by RNA3 and/or RNA5.     

Incidence of viruses in <Emphasis Type="Italic">Alstroemeria</Emphasis> plants cultivated in Japan and characterization of <Emphasis Type="Italic">Broad bean wilt virus-2, Cucumber mosaic virus</Emphasis> and <Emphasis Type="Italic">Youcai mosaic virus</Emphasis>

Alstroemeria plants were surveyed for viruses in Japan from 2002 to 2004. Seventy-two Alstroemeria plants were collected from Aichi, Nagano, and Hokkaido prefectures and 54.2% were infected with some species of virus. The predominant virus was Alstroemeria mosaic virus, followed by Tomato spotted wilt virus, Youcai mosaic virus (YoMV), Cucumber mosaic virus (CMV), Alstroemeria virus X and Broad bean wilt virus-2 (BBWV-2). On the basis of nucleotide sequence of the coat protein genes, all four CMV isolates belong to subgroup IA. CMV isolates induced mosaic and/or necrosis on Alstroemeria. YoMV and BBWV-2 were newly identified by traits such as host range, particle morphology, and nucleotide sequence as viruses infecting Alstroemeria. A BBWV-2 isolate also induced mosaic symptoms on Alstroemeria seedlings.     

Characteristics of a <Emphasis Type="Italic">Plasmopara angustiterminalis</Emphasis> isolate from <Emphasis Type="Italic">Xanthium strumarium</Emphasis>

Leaves of Xanthium strumarium infected with downy mildew were collected in the vicinity of a sunflower field in southern Hungary in 2003. Based on phenotypic characteristics of sporangiophores, sporangia and oospores as well as host preference the pathogen was classified as Plasmopara angustiterminalis. Additional phenotypic characters were investigated such as the size of sporangia, the number of zoospores per sporangium and the time-course of their release. Infection studies revealed infectivity of the P. angustiterminalis isolate to both X. strumarium and Helianthus annuus. Inoculation of the sunflower inbred line, HA-335 with resistance to all known P. halstedii pathotypes, resulted in profuse sporulation on cotyledons and formation of oospores in the bases of hypocotyls. Infections of sunflower differential lines often led to damping-off. Molecular genetic analysis using simple sequence repeat primers and nuclear rDNA sequences revealed clear differences to Plasmopara halstedii, the downy mildew pathogen of sunflower.     

Biofilm formation and resistance to bactericides of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">theae</Emphasis>

Biofilm-grown cells of Pseudomonas syringae pv. theae (P.s.theae) wild-type strain K9301 on abiotic surface had remarkable resistance to kasugamycin in comparison to planktonically grown cells; however, the biofilm-grown cells of K9301 had the same sensitivity to copper sulfate. Because both the lesser biofilm-forming strain K9301S3 and enhanced biofilm-forming strain K9301-6 also had remarkable biofilm resistance to kasugamycin just as K9301 did and because epigallocatechin gallate, which enhanced biofilm formation of P.s.theae, had no effect on biofilm resistance to kasugamaycin, the degree of biofilm formation was not correlated with the antibiotic susceptibilities. In addition, K9301 and K9301S3 had less sensitivity to kasugamycin but had high sensitivity to copper sulfate on nonwounded leaf surfaces. These results indicate a possibility that the mechanism of P.s.theae biofilm resistance to bactericide functions on both abiotic and nonwounded leaf surfaces.     

Virulence and diversity of <Emphasis Type="Italic">Blumeria graminis</Emphasis> f.sp. <Emphasis Type="Italic">hordei</Emphasis> in East China

Four hundred and sixty-one isolates of Blumeria graminis f.sp. hordei were obtained from eight populations occurring on cultivated barley (Hordeum vulgare) at four geographically distant locations in China during 2003 and 2004. Their virulence frequency was determined on 30 differential lines. No isolate was virulent on differential lines possessing the resistance genes Mla1, Mla3, Mla6, Mla7, Mla9, Mla12, Mla13, Mlat, Mlg, Mla10, Mla22, Mla23, Mlp1, Ml(N81) and Mlmw. Virulences to the first nine resistance genes are prevalent in Europe and constitute the main part of genetic distance between Chinese and European populations. Conversely, no isolate was avirulent on the differential lines possessing the genes Mla8 and Ml(Ch). The frequencies of isolates overcoming the genes Mla2, Mla11, Mlk1 and Mlk2 were .4–9.3%, and frequencies of isolates overcoming the genes Mlh, MlLa, Ml(Bw), Mlra, Ml(Ru2), mlw, MlGa, MlWo and Mlnn ranged from 18.2% to 98.7%. Based on reactions of differential lines possessing the genes Mlk1, Mlh, MlLa, Ml(Bw), Mlra and Ml(Ru2), pathotypes were identified and diversity parameters calculated. Eleven of 22 detected pathotypes were found in both years and comprised 94.6% of isolates. Generally, the populations from different locations in 1 year were more closely related than populations collected from the same locations in different years. Complete effectiveness of the resistance genes, for which no corresponding virulences were found, will allow Chinese breeders to access many modern European barley cultivars that are fully resistant to powdery mildew in China, including those possessing the non-host resistance gene mlo.     

A new <Emphasis Type="Italic">Rhizoctonia</Emphasis> sp. closely related to <Emphasis Type="Italic">Waitea circinata</Emphasis> causes a new disease of creeping bentgrass

Isolates of an unidentified Rhizoctonia sp. (UR isolates) were obtained from creeping bentgrass and Kentucky bluegrass with reddish brown sheath and foliar rots. Because the UR isolates anastomosed with isolates of three varieties of Waitea circinata (var. oryzae, var. zeae, and var. circinata), colony morphology, hyphal growth rate at different temperatures, pathogenicity, sequence analysis of the internal transcribed spacers (ITS) region of ribosomal RNA genes (rDNA) were compared. The colony color of mature UR isolates was distinct from isolates of the other three varieties of W. circinata. In pathogenicity tests on creeping bentgrass, the severity of the disease caused by UR isolates was significantly higher than that caused by the three varieties of W. circinata. Sequence similarities of the rDNA-ITS region between UR isolates and between isolates within each variety were high (97–100%), but they were lower among isolates from UR and the varieties of W. circinata (88–94%). In a phylogenetic tree based on the rDNA-ITS sequences, UR isolates formed a cluster separate from each of the clusters formed by the three varieties of W. circinata. These results indicate that the UR isolates clearly differ from the three varieties of W. circinata. We therefore propose that the UR isolates be classified as new Rhizoctonia sp. that are closely related to W. circinata and that the disease on creeping bentgrass should be called Waitea reddish-brown patch disease (Sekikasshoku-hagusare-byo in Japanese).