Phylogenetic diversity of <Emphasis Type="Italic">Calonectria ilicicola</Emphasis> causing Cylindrocladium black rot of peanut and red crown rot of soybean in southern China

Calonectria ilicicola Boedijin & Reitsma (anamorph: Cylindrocladium parasiticum Crous, Wingfield & Alfennas) is an important pathogen worldwide, which causes Cylindrocladium black rot (CBR) in peanut (Arachis hypogaea L.) and red crown rot (RCR) in soybean [Glycine max (L.) Merr.]. We isolated the CBR and RCR pathogens from heavily diseased peanut and soybean fields in southern China and assessed their pathogenicity. Two inoculation methods were applied separately to evaluate the pathogenicity of different C. ilicicola strains on peanut cultivar Yueyou 13. Our results indicate that the Chinese C. ilicicola strains exhibited a range of virulence on peanut, with strains of soybean origin exhibiting a weak virulence relative to strains isolated from peanut. Multilocus sequence type analysis indicates that the C. ilicicola strains partitioned into two distinct clades, which were heavily structured based on geographical origin. Phylogenetic results demonstrated that the origins of C. ilicicola in southern China were multiple. This study also revealed that the backgrounds of CBR pathogens may be different from those of RCR pathogens.     

A simple method for long-term cryopreservation of <Emphasis Type="Italic">Calonectria ilicicola</Emphasis> on barley grains

We developed a simple method for long-term preservation of the soybean red crown rot fungus, Calonectria ilicicola, using barley grains. Autoclaved barley grains were inoculated with the fungus, then incubated at 25°C for 1 month. After incubation, grains were dried to approximately 3% moisture content, and stored at 4, −20, or −80°C for 3 years. C. ilicicola preserved on barley grains at −80°C remained viable without any change in mycelial growth and virulence. These results showed that C. ilicicola can be successfully cryopreserved for extended periods on barley grains at −80°C. We also confirmed that cultures preserved on barley grains are suitable for direct use without further manipulation as inocula in pathogenicity tests.     

Pathogenicity studies in avocado with three nectriaceous fungi,Calonectria ilicicola,Gliocladiopsis sp. and Ilyonectria liriodendri

Calonectria ilicicola, Gliocladiopsis sp. and Ilyonectria liriodendri were isolated from diseased roots of young avocado trees. Pathogenicity studies with seedlings of three avocado cultivars, Velvick, Hass and Reed, demonstrated that Calonectria ilicicola is a severe root rot pathogen, reducing the biomass of healthy roots, and reducing plant height over time. Calonectria ilicicola was re‐isolated from diseased roots. Ilyonectria liriodendri and Gliocladiopsis sp. were not pathogenic and plant height was increased after Gliocladiopsis sp. amendment compared to all other treatments in trials with cvs Velvick and Hass.     

New Selective Medium for Isolation of Burkholderia glumae from Rice Seeds

A new selective medium for Burkholderia glumae was developed, which has a simpler composition and greater selectivity compared to Tsushima's S-PG medium currently used. This selective medium, designated CCNT, contains 2 g of yeast extract, 1 g of polypepton, 4 g of inositol, 10 mg of cetrimide, 10 mg of chloramphenicol, 1 mg of novobiocin, 100 mg of chlorotharonil and 18 g of agar in 1000 ml of distilled water, and is adjusted to pH 4.8. B. glumae produced a yellowish white colony with a diffusible yellow pigment on CCNT medium, which was distinguishable from other bacterial species when incubated at 41°C for 2 to 4 days. On CCNT medium, B. glumae was detected at a rate similar to that on S-PG medium in rice seeds, while other microorganisms were detected at a much lower rate. Received 16 September 1999/ Accepted in revised form 4 January 2000     

Time,concentration and temperature relationships for phosphine activity in tests on diapausing larvae of Ephestia elutella (Hübner) (Lepidoptera: Pyralidae)

The activity of phosphine against diapausing larvae of Ephestia elutella was shown to depend on several different interactions of duration of exposure, temperature and gas concentration. Using the model proposed by Knight, (C? C₀) × (t? t₀) = k, a constant for mortality, the minimum effective concentration C₀ was raised and the minimum effective exposure t₀ extended as temperatures were increased. The effect of temperature on these thresholds was reduced at higher mortality levels. A range of concentration levels, influenced by temperature, elicited an increase in tolerance at the LD₉₉ level over that evident at higher or lower concentrations. These concentration ranges were 0.9–2-1 mg litre-¹at 25°C,0.7–2.3 mg litre-¹ at 20°C and 0.1–1.5 mg litre-¹ at 15°C. A mathematical model was fitted to relate concentration and time thresholds, mortality and temperature. At the LD₅₀ level, values for t₀ ranged from 2.5 h at 15°C to 5.0 h at 25°C. C₀ values increased from about 0.6 to 7.0 μg litre-¹ over this temperature range. At the LD₉₉ level, t₀ values ranged from 21 to 28 h and C₀ values from 11 to 19 μg litre-¹. Phosphine appeared most effective at each temperature at concentrations from about 40 to 60–100 μg litre-¹.     

The relationship between brain levels of cismethrin and bioresmethrin in female rats and neurotoxic effects

The oral toxicity of 5-benzyl-3-furylmethyl-(1R, cis)-chrysanthemate (cismethrin) to female rats decreased as their environmental temperature was raised. Acute oral LD₅₀ values increased from 157 mg/kg at 4°C to 197 mg/kg at 20°C and to > 1000 mg/kg at 30°C. Cismethrin was much more toxic given intravenously when the LD₅₀ was 4.5 mg/kg. This value did not change at different environmental temperatures. Irrespective of the environmental temperature, or route of adminstration, following the respective LD₅₀'s cismethrin caused tremors in rats when brain levels of 0.5–1.0 μg/g were reached and, at death, brain concentrations were 3.9–5.1 μg/g. These results suggested that the accumulation of cismethrin by the brain could be used as a model for the nervous system as a whole. The isomeric 5-benzyl-3-furylmethyl-(1R, trans)-chrysanthemate (bioresmethrin) was about 50 times less toxic to rats than cismethrin. After an intravenous LD₅₀, tremors started when brain concentrations were 4–5 μg/g. At death, brain levels were 25–35 μg/g. Plasma esterases were about equally active in hydrolysing cismethrin and bioresmethrin, whereas liver microsomal esterases hydrolyzed bioresmethrin over 10 times more rapidly than cismethrin. It is suggested that the lower toxicity of bioresmethrin is not only due to its faster metabolism but to an intrinsically lower toxicity at the critical site of action in the nervous system.     

Überleben von Ralstonia solanacearum Biovar 2 (Rasse 3), dem Erreger der Schleimkrankheit der Kartoffel, in Boden und auf verschiedenen Materialien (Mikrokosmos)

Microcosm studies were carried out to test the survival of Ralstonia solanacearum biovar 2 (race 3) in soil at the permanent wilting point (wp) water content and at field capacity (fc) water content and on various material. Soils were placed at permanent ?5°C, 4°C, 15°C and 20°C and weekly fluctuating ?10/0/+10°C and the material at 5, 15 °C, 20°C with relative humidity (rh) uncontrolled or at constant 10% or 90%. In soil, survival was clearly dependent on temperature independent of water content. At 20°C Ralstonia solanacearum could be reisolated up to 364 days, at 15°C up to 290 days, at 4°C up to 209 days and at fluctuating temperatures (?10/0/+10°C) only up to 18 days. The lower the temperature, the more the population declined. At 15°C and 20°C appr. 10⁷ cfu/g soil were detected after 100 days, whereas at ?5°C only 10² cfu/g soil were detected after only 18 days. The pathogen was longer detectable in sandy-clay loam than in lighter sandy soil. It could be longer reisolated at wilting point and the populations did not decline as rapidly as at field capacity. Ralstonia solanacearum could best survive on material surfaces like rubber, plastic and varnished metal with maximum survival of 40 days at 5°C and 10% rh. In general there is a low risk of Ralstonia solanacearum overwintering under European climatic conditions when the fields are cleared of plant debris and the soil is frozen. Contamined material surfaces pose the risk of pathogen transmission to healthy tubers.     

Cylindrocladium brown leaf spot on Howea belmoreana caused by Calonectria ilicicola (anamorph: Cylindrocladium parasiticum) in Japan

Brown leaf spot disease caused by Cylindrocladium was found on Howea belmoreana on Hachijojima Island, Tokyo, Japan, in December 2001. Typical symptoms were incited after artificial inoculation. A culture of white mycelia, isolated from leaf spot symptoms, produced reddish perithecia of a nectriaceous fungus. Based on morphological and molecular analyses, this fungus was identified as Calonectria ilicicola (anamorph: Cylindrocladium parasiticum). Pathogenicity of this fungus on five plants cultivated on Hachijojima Island was confirmed by artificial inoculation. This report is the first on Cylindrocladium brown leaf spot of H. belmoreana caused by C. ilicicola (anamorph: Cy. parasiticum).     

Low temperatures enhance winter wilt of pepper plants caused by Pythium sp.

Pepper is the main vegetable crop grown in the Arava region of southern Israel. It is grown in the winter in nethouses and greenhouses. Low temperature wilt of mature pepper plants has been known for years in this region. The incidence of plant wilting was usually low when the soil was pretreated with methyl bromide. In recent years methyl bromide usage has been banned and disease incidence has increased. The causal agent of this phenomenon was unknown until the current study. Pythium sp. was the most common microorganism genus isolated from wilted plant roots. Young pepper plants were artificially inoculated with Pythium isolated from wilted plants and maintained at temperatures of 20°, 14°, 10.5° and 8.6°C. Significant wilting was observed in plants grown at 8.6°C, with symptoms starting 2?weeks after inoculation. At 10.5°C wilting developed more slowly and inoculated plants maintained at 14° and 20°C did not exhibit any wilting symptoms. The unique variation in sporangium morphology and the sequence of the ribosomal internal transcribed spacer (ITS) suggest that a new species of Pythium is involved. The fungicide metalaxyl-M was found effective in controlling the disease in pot experiments. The relationship between low temperatures and high disease incidence can explain the high disease incidence in the Arava Valley of Israel during the cold winters of 1999?C2000, 2004?C2005 and 2006?C2007.     

Characterization of cytochrome b from European field isolates of Cercospora beticola with quinone outside inhibitor resistance

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar disease of sugar beet worldwide. Control strategies for CLS rely heavily on quinone outside inhibitor (Q_OI) fungicides. Despite the dependence on Q_OIs for disease control for more than a decade, a comprehensive survey of Q_OI sensitivity has not occurred in the sugar beet growing regions of France or Italy. In 2010, we collected 866 C. beticola isolates from sugar beet growing regions in France and Italy and assessed their sensitivity to the Q_OI fungicide pyraclostrobin using a spore germination assay. In total, 213 isolates were identified with EC₅₀ values greater than 1.0???g?ml^?1 to pyraclostrobin, all of which originated from Italy. To gain an understanding of the molecular basis of Q_OI resistance, we cloned the full-length coding region of Cbcytb, which encodes the mitochondrial Q_OI-target enzyme cytochrome b in C. beticola. Cbcytb is a 1,162-bp intron-free gene with obvious homology to other fungal cytb genes. Sequence analysis of Cbcytb was carried out in 32 Q_OI-sensitive (<0.080???g?ml^?1) and 27 Q_OI-resistant (>1.0???g?ml^?1) isolates. All tested Q_OI-resistant isolates harboured a point mutation in Cbcytb at nucleotide position 428 that conferred an exchange from glycine to alanine at amino acid position 143 (G143A). A PCR assay developed to discriminate Q_OI-sensitive and Q_OI-resistant isolates based on the G143A mutation could detect and differentiate isolates down to approximately 25?pg of template DNA. Microsatellite analyses suggested that Q_OI resistance emerged independently in multiple genotypic backgrounds at multiple locations. Our results indicate that Q_OI resistance has developed in some C. beticola populations in Italy and monitoring the G143A mutation is essential for fungicide resistance management in this pathosystem.     

Cultural characteristics of Sporisorium sorghi and detection of the pathogen in plant tissue by microscopy and polymerase chain reaction

Despite the economic importance of covered kernel smut of sorghum (Sporisorium sorghi) in many African states and other parts of the world, only limited information is available on laboratory cultivation methods for this fungus and techniques for its diagnosis in plant tissue. The current paper describes laboratory and greenhouse experiments performed with field material of S. sorghi. When intact sori were kept at 5°C, 80% of the spores germinated even after 24?months of storage. Spore germination on agar medium and production of mycelial dry weight in still culture were highest between 20° and 35°C, with a peak at 30°C. Both showed a steady increase from pH 4.5 to pH 7.5, followed by a decline at pH 8.5 and 9.5. In shake culture in different broth media the addition of 0.3% peptone from soybean caused an increase in fungal growth compared with the media alone. Of the media tested, mycelial production was highest in malt dextrose broth supplemented with peptone. When cultivated on different agar media, the morphology of single spore isolates differed both among isolates and depending on the agar medium. In greenhouse experiments, five short, early maturing sorghum breeding accessions proved to be partially or fully resistant to covered kernel smut. Among the plant materials tested, cv. ??Dorado?? appeared to be the one best suited for greenhouse experiments with covered kernel smut. By microscopy of hand-cut sections stained with trypan-blue, hyphae of S. sorghi were seen in apical buds and in nodes of young sorghum plants. Diagnostic PCR amplified a 903?bp element comprising the internal region of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoding gene and enabled the detection of S. sorghi in both nodes and apical buds of infected sorghum seedlings. Both techniques, i.e., microscopy and diagnostic PCR, have the potential to be used in studies for the identification of effective sorghum seed treatments already at the seedling stage.     

降解菌2N3对被氯嘧磺隆污染土壤的生物修复

在实验室条件下,研究了高效降解菌2N3(克雷伯氏菌属,Klebsiella sp.)对被氯嘧磺隆污染土壤的修复作用及其影响因素。当土壤中氯嘧磺隆的添加浓度为20 mg/kg,每 1克土壤中2N3的接菌量为1×106个菌体时,第30 d时土壤中氯嘧磺隆的降解率为84.6%,对照仅为13.4%;相同2N3接菌浓度下,当土壤中氯嘧磺隆浓度为100 mg/kg时,其降解率为31.1%。以小麦、玉米、黄瓜为供试作物,在土壤中施加 20 mg/kg的氯嘧磺隆, 当每 1克土壤中2N3的接菌量为1×106个 菌体时,小麦、玉米、黄瓜的出苗率分别为85%,82%和79%,且处理组株高高于对照,表明降解菌2N3具有明显减轻氯嘧磺隆药害的作用。研究表明,人工接种降解菌2N3可提高土壤中氯嘧磺隆的降解率,有效降低其在土壤中的残留。     

Effects of triazoles on fungi: I. Growth and cellular permeability

Effects of the triazoles propiconazol (CGA-64250), etaconazol (CGA-64251), and triadimefon, which are inhibitors of the C-14 demethylation of lanosterol, on the growth of 14 fungal species and cell permeability of Taphrina deformans were determined. With the exception of the Mucor spp. tested, growth was inhibited approximately 50% by these inhibitors at 0.073 to 5.0 μg/ml medium. Propiconazol appeared to be fungistatic rather than fungicidal against Sclerotium rolfsii, whereas T. deformans showed only limited growth after pretreatment with 50 μg/ml. Temperature (15 to 31°C) did not affect the inhibitory action of propiconazol toward Rhizopus stolonifer, and the growth response by T. deformans to the inhibitor was essentially the same at medium pH values in the 5.4 to 7.0 range. Although the flux of some substances across the cell membrane was altered in cells grown in medium containing the ED₅₀ concentration of propiconazol, this does not appear to be a principal factor in growth inhibition by this compound. The modest changes in cell permeability appeared to be due to inhibitor-induced metabolic changes (e.g., C-14 demethylation inhibition) rather than a direct affect on the membrane by the inhibitor.     

Development of Real-Time PCR for in wood-detection of Ceratocystis platani, the agent of canker stain of Platanus spp.

Ceratocystis platani is a quarantine fungal pathogen agent of canker stain, a destructive disease affecting Platanus. Despite its diagnosis being critical for disease control, there is still no effective diagnostic tool as all known mycological and biological detection assays are problematical. In this study we developed highly effective Real-Time PCR methods based on the use of an intercalating dye, EvaGreen, and on a Taqman probe. We designed primers and probe on the internal transcribed spacer (ITS) region of rDNA, and used them for the amplification and detection of a 95?bp C. platani amplicon. Inference of standard curves revealed that both Real-Time procedures have similar and high values of amplification efficiency when applied to a range of templates, e.g. genomic fungal DNA and DNA extracted from diseased wood. The methods were sensitive with a detection limit of 10?fg???l^?1? C. platani genomic DNA. They were specific as they did not yield any detection signal when applied to non-target fungal taxa colonizers of Platanus wood. Reliability was demonstrated through the positive detection of a collection of C. platani isolates and of wood samples collected from naturally infected trees. Robustness was positively verified through detection on artificially infected trees, which were tested at different times after death, up to 27?months. Generating a standard curve with a target-amplicon-containing plasmid enabled an absolute quantification and a comparison between the discoloured wood of resistant and susceptible genotypes. The importance of the methods for studies on pathogen epidemiology and host resistance is also discussed.     

Cry1Ac和Cry2Ab蛋白对大草蛉生长发育及酶活力的影响

为明确转Cry1Ac和Cry2Ab基因棉花对大草蛉的影响,运用Bt蛋白与人工饲料混合的方法,以大于转基因棉花叶片中蛋白含量20倍的剂量饲喂大草蛉初孵幼虫,初步研究了Cry1Ac和Cry2Ab对大草蛉生物学参数和消化酶、解毒酶活性的影响。结果表明：取食含Bt蛋白饲料的大草蛉幼虫的发育历期、体重、蛹重、成虫体重、羽化率等生物学参数与对照相比均没有显著差异;在大草蛉幼虫体内可以检测到含量较高的Cry1Ac和Cry2Ab蛋白,分别为974.92~1 282.39 ng/g鲜重和5 592.62~6 082.92 ng/g鲜重,而在大草蛉成虫体内检测到的Cry1Ac和Cry2Ab蛋白含量非常低,分别为0.29~0.39 ng/g鲜重和50.34~56.71 ng/g鲜重;取食含Bt蛋白的饲料对大草蛉幼虫的类胰蛋白酶、类胰凝乳蛋白酶、氨肽酶和谷胱甘肽-S-转移酶活性有一定的影响,但对大草蛉成虫影响与对照差异不显著。表明大草蛉取食含Bt蛋白的人工饲料后,虽然体内可以检测到一定含量的Bt蛋白,但对大草蛉的生长发育并没有显著的直接不利影响。     

Elimination of Grapevine rupestris stem pitting-associated virus (GRSPaV) from two Vitis vinifera cultivars by in vitro chemotherapy

The grapevine (Vitis vinifera L.) cultivars ‘Agiorgitiko’ and ‘Malagouzia’, naturally infected with Grapevine rupestris stem pitting-associated virus (GRSPaV), were subjected to in vitro chemotherapy using the antiviral inosine 5′-monophosphate dehydrogenase inhibitors tiazofurin (TR), ribavirin (RBV) and mycophenolic acid (MPA). The chemotherapy lasted 80 days and was carried out as two consecutive treatments. Severe phytotoxicity, estimated after 40 days of culture, was observed in drug-treated explants, especially when high doses of TR were used. Phytotoxicity exhibited a cultivar- and chemical compound-dependent profile. The virus eradication status of the survived plantlets was determined by nested RT-PCR using total RNA templates, after 80 days of drug treatment and one year later, after the passage of one dormancy period, in potted plants grown in a greenhouse. Data indicated that the highest GRSPaV elimination in ‘Agiorgitiko’ was obtained with 10 μg? ml^?1 TR, 30 μg ?ml^?1 RBV and 20 μg? ml^?1 MPA. The eradication rates were lower in the case of ‘Malagouzia’, where the highest ones were achieved after treatments with 15 μg ml^?1 TR and 80 μg ml^?1 MPA. This is the first report on GRSPaV elimination in grapevine following treatment with antiviral compounds, which could provide an alternative to the traditional methods of virus eradication through meristem culture and thermotherapy.     

A pollen test to detect ACCase target‐site resistance within Alopecurus myosuroides populations

This study was conducted to determine a suitable medium for in vitro germination of Alopecurus myosuroides pollen and to develop a reliable test for the rapid screening of ACCase target site‐resistant plants within populations. The assay is based upon germination of pollen in a medium supplemented with ACCase inhibitors. A 0.25% agar medium, containing 200 mg L^–1 CaNO₃, 100 mg L^–1 H₃BO₃, 200 g L^–1 sucrose, was selected as a suitable medium for in vitro pollen germination. At 25 °C, this medium supported a mean germination rate of 85% within two hours. Plants highly resistant (Rh) to aryloxyphenoxypropionate (APP), owing to the expression of an insensitive ACCase, were found to express this resistance in their pollen. In contrast, plants moderately resistant (Rm) to APP herbicides, owing to an enhanced capacity to detoxify herbicides, did not exhibit this resistance in their pollen. Concentrations of 120 μM fenoxaprop and 1000 μM clodinafop were selected as the best for reliable discrimination of the target‐site‐resistant biotypes. At these concentrations there was more than 50% germination of the Rh pollen grains whereas less than 10% of the S and Rm pollen grains germinated. This test, using haploid material, may also permit distinction between homozygous‐ and heterozygous‐resistant individuals.     

Identification of some Fruit Characteristics in Wild Bilberry (<Emphasis Type="Italic">Vaccinium myrtillus</Emphasis> L.) Accessions from Eastern Anatolia

Some important physicochemical and bioactive characteristics of disease free 10 wild grown bilberry (Vaccinium myrtillus) accessions were evaluated. External and internal fruit quality was assessed by standard parameters (fruit weight, fruit color, fruit firmness, soluble solids, pH and total acidity) and bioactive contents (total phenolics, total anthocyanins, total antioxidant capacity and, vitamin C) in fruit were also determined. The commercial grown northern higbush blueberry, Vaccinium corymbosum cv. Bluecrop also included in the study to make comparision with bilberry samples. The highbush blueberry cv. Bluecrop had distinctive external fruit characteristics, such as bigger and more attractive fruits. However, the wild grown bilberry accessions showed interesting characters in mesocarp, such as high total phenolic content, total anthocyanin and total antioxidant capacity. Total phenolic and total anthocyanin content was 327?mg gallic acid equivalent and 142?mg of cyanidin-3-glucoside equivalent in 100?g fresh fruit in cv. Bluecrop while it was between 576–624?mg gallic acid equaivalent and 296–324?mg of cyanidin-3-glucoside equivalent in 100?g fresh fruits of bilberry accessions. Moreover, wild accessions approximately had 2 folds higher antioxidant capacity than cv. Bluecrop. Results suggested that bilberry accessions may serve as a source of desirable genes to develop improved varieties that respond to the new needs of the market.     

芒果炭疽病菌的抑菌植物筛选及肉桂油抑菌活性测定

以芒果炭疽病菌抗药性菌株、敏感菌株为研究材料,用生长速率法筛选出了一批高效抑菌植物,大部分植物的丙酮提取物对芒果炭疽病菌有不同程度的抑制作用,其中:肉桂、丁香、藿香的提取物活性最强,在干物质质量浓度为0.025 g/mL下抑制率达100%;芒果炭疽病菌抗药性菌株对肉桂等植物提取物无明显抗性;肉桂油对芒果炭疽病菌敏感菌株(ZJS)和抗药性菌株(JZR)的抑制中浓度(EC50)分别为98.9和71.2 mg/L,而肉桂醛对ZJS和ZJR的EC50值分别为20.7和20.2 mg/L。