White Hyphae Research Articles

First Report of Sclerotium rolfsii Causing Southern Blight of Bletilla Orchid in China

Bletilla striata, known as Chinese ground orchid, is economically important because of its medicinal and ornamental properties around the world, especially in China. The wild population of B. striata is decreasing, it was listed in appendix II (species that may be threatened or extinct) of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) (CITES 2013). Therefore, farm-based cultivation of B. striata is emerging to meet the demands of the industry. In June 2016, disease symptoms similar to orchid southern blight (Punja 1985) were observed in a B. striata cultivation field (N30°21′27.37″, E112°19′58.37″) in Jingzhou, Hubei Province, China. In a follow-up survey, the disease was also found in nearby counties and provinces. Disease incidence reached 25% in the most severely affected field. Symptoms first appeared at the junction of the stem base and the soil, as necrotic dark brown stem lesions. Additional symptoms included leaf yellowing and wilting, root rot, and death during warm and humid weather. White mycelium and tan to brown sclerotia were found on the stems and roots of plants in the field and plants incubated in the lab. To isolate the pathogen, tissue from plant samples collected from a nursery (Orchid Biotechnology, Jingzhou, China) was surface sterilized with 2% sodium hypochlorite (NaOCl) for 3 min and then rinsed and incubated on potato dextrose agar medium with 50 μg/ml of ampicillin and rifampin. After 7 days of incubation at 28°C, sclerotial primordia developed followed by sclerotia approximately 2 mm in diameter after 10 days. No spores were observed. Total genomic DNA of isolate BH-2 was extracted with the cetyltrimethylammonium bromide method and then amplified and sequenced with fungal ribosomal primers ITS1/ITS4 (White et al. 1990). The length of the amplicon was 683 bp (GenBank accession no. KY606616), 99% similarity to Athelia rolfsii (teleomorph) isolate ATCC201126 (accession no. AF499018). Phylogenetic analysis of the internal transcribed spacer sequences with the neighbor-joining method in MEGA6 (Pennsylvania State University, University Park, PA) showed BH-2 was clustered with six Sclerotium rolfsii isolates selected from NCBI database (accession nos. AF499018, JN081867, JN241565, GQ3585118, JF966208, and KJ002764). Therefore, isolate BH-2 was identified as S. rolfsii. To produce inoculum for Koch’s postulates, autoclaved oat grain was inoculated with BH-2 then incubated at 30°C for 10 days. Infected oat grains were placed near the roots of 90-day-old healthy seedlings (n = 3), and sterilized oat grain was used as a control (n = 3). All seedlings were placed in sealed plastic bags with air valves and grown in a greenhouse (25 ± 2°C, relative humidity 65%). Water-soaked streaks and white hyphae were observed after 2 to 3 days. Symptoms typical of southern blight, necrotic stems, and leaf lesions were observed after 9 days. Reisolation was performed with surface-sterilized tissue from symptomatic seedlings, and isolates were confirmed as S. rolfsii based on morphological and molecular characters. To the best of our knowledge, this is the first report of S. rolfsii causing southern blight of Bletilla spp. in China.

Papilla formation, defense gene expression and HR contribute to the powdery mildew resistance of the novel wheat line L699 carrying Pm40 gene

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive wheat diseases worldwide and causes severe yield losses in most wheat-growing areas. POWDERY MILDEW (Pm) genes are responsible for wheat resistance to Bgt. Recently, a wheat line L699 carrying a novel Pm40 gene which was originated from Elytrigia inermedium, was developed and showed high resistance to Bgt; however, the resistance mechanism of L699 is still unclear. In current study, a range of defense responses were examined in wheat line L699 inoculated with Bgt, using a highly susceptible cultivar CN26 without Pm40 gene as a control. We found that high levels of reactive oxygen species (ROS) were rapidly accumulated and abundant papilla were formed at the infection sites where Bgt conidiospores germinated with abnormal morphology at a high frequency on L699 compared to CN26. Local hypersensitive cell death and defense genes in the salicylic acid (SA) and jasmonic acid (JA) signaling pathways were then rapidly induced in L699. As a consequence, L699 exhibited no typical disease symptoms, whereas CN26 were fully covered by white hyphae and conidia. These data provide evidence that for the resistance of the wheat line L699 with the dominant gene Pm40 against Bgt, early ROS accumulation and papilla formation acts as dominant penetration resistance, leading to morphological abnormality of Bgt, when defense gene expression in SA and JA signaling and HR response enhance the post-penetration resistance by arresting onset of Bgt.

ISOLASI DAN KARAKTERISASI Beauveria bassiana SEBAGAI FUNGI ANTI HAMA

Development of biological potential could to be a solution of pest problems and environmental damage by pesticides. One of the organisms that are currently often be used for biopesticides is the entomopathogenic fungi such as Beauveria brassiana. This study aim to isolate, characterize of pest-resistant fungi and apply it to some agricultural insect pests in vitro. Samples which used were planthoppers, aphids, grasshoppers and isolated fungi Beauveria bassiana from Biogen Laboratory. The research was conducted in Soil and Environmental Biotechnology Laboratory, Dramaga, Bogor. Isolation method with Insect Bait Methode. Isolation and characterization of pest-resistant fungi that are planthoppers and aphids show the result that the great possibilities are the Beauveria bassiana fungus, that is clearly visible from the obtained physical characteristics, the white and sealed hyphae and conidia round oval. In addition, the fungi which used is a pathogenic fungi on the pest of aphids, planthoppers, and grasshoppers.

Eksplorasi dan identifikasi Jamur Patogen Serangga di Rhizosfer Lahan Kering Kabupaten Malang

This research was conducted by exploring insect pathogen in rhizosphere of dry field of sugar cane of Malang Regency. The purpose of this research is to study the diversity of isolate insect pathogen in rhizosphere of sugar cane plant in dry land of Malang Regency. Exploration activities of insect pathogens are carried out by isolating insect pathogens in dry land of sugarcane plant rhizosphere. Rizosphere soil sampling method using composite sampling technique is by taking diagonal soil samples at five points as deep as 5-10 cm, as much as 200 g at each sample point and isolation of insect pathogen type fungus using baiting method. The soil samples from the field were placed evenly over the petri dish. The soil above the petri dish is moistened and then the larvae of T miltor are placed for incubation for 48 hours. After 48 hours of dead larvae there will be a sign of an insect pathogenic fungus in the form of white hyphae on the surface of the T milter body. The marked larvae were then cultured on PDA media to be purified and identified morphologically, by looking at the color of hyphae. Observation miscroscopically using Burnet and Hunter determinations. Exploration and identification results found four specimens of fungi with Aspergilus sp species, Trichoderma sp, Beuveria sp, and Metharizium sp.

Detection of fungi from rice black bug Paraeucosmetus pallicornis Dallas (Hemiptera: Lygaeidae) and inhibition with crude extract of Calatropis gigantea (Asclepiadaceae)

Rice black bug (P. palicornis) is one of the pests that attack the rice plants in the generative phase that causes the rice easily destroyed when milled and tasted bitter after cooking hence reduces the quality and quantity of rice. The bitter taste in rice may be due to the fungus associated with rice black bug. The aimed of this research was to detect the associated fungi with rice black bug P. pallicornis using some sterilization methods and inhibition with of leaf crude extract of C. gigatea. Detection of fungi from P. pallicornis was conducted using three sterilization methods and control (without sterilization) namely: (1) sterilization with aquades + alcohol 70% (5, 10, 15 and 20 times dipping) + aquades; (2) aquades + alcohol 70% (10 and 20 times dipping); (3) Aquades + alcohol 90 %+ NaCl 0.5 % +alcohol 90 % + aquades. Inhibition of fungi from P. pallicornis with crude extract of C. gigantea obtained by maceration method and then made some concentration to see the effect of its inhibition on the fungi associated with the P. pallicornis. The results showed that without sterilization, four microbe were obtained: Gliocladium sp., Aspergillus sp., black hyphae fungus and white hyphae fungus, sterilization method of Aquades + alcohol 70% with 5 times dipping in alcohol obtained Gliocladium sp., 10 and 20 times dipping found Aspergillus sp. and Gliocladium sp and 15 times dipping found Aspergillus sp. Sterilization with 10 and 20 times dipping in alcohol 70% then washing 2 times with aquades found Gliogladium sp. and Aspergillus sp. Sterilization with Aquades + alcohol 90 % + NaCl 0.5% + alcohol 90% + aquades found Gliocladium sp. Crude extract of C. gigantea had the potential to inhibit fungi Aspergillus sp. and Gliocladium sp. from rice black bug.

Density, Viability Conidia And Symptoms of Metarhizium anisopliae infection on Oryctes rhinoceros larvae

M. anisopliae is parasitic fungus on insect pests; it is used as a biocontrol agent. M. anisopliae can be propagated on maize or rice substrate. M. anisopliae is currently sold in the form of kaolin powder formulations. Before it is used to check the density, viability and pathogenicity of M. anisopliae. However the problem is the kaolin powder very soft, so it difficult to distinguish between kaolin and conidia. This article gives information on how to calculate conidia density, viability and symptoms of M. anisopliae infection on Oryctes rhinoceros larvae. The study was conducted in the laboratory to determine the density and viability. The pathogenicity testing was done using pots. The Pot is containing soil substrate mixed with M. Anispoliae and ten tails O. Rhinoceros larvae per pot. The results showed that the density of M. anisopliae conidia was 1.81 x 108 conidia mL-1 and the viability was 94% within 24 hours. The larval mortality began to emerge in the 1st week, and all larvae died at the sixth week. The symptom of M. anisopliae infection on Oryctes rhinoceros larvae, there was a black spot on the larval integument. The larvae movements become slow and poor appetite; it will die within 3-7 days. The larvae die hard, and the white hyphae grow on the body surface that turns green.

FIRST REPORT OF MICROSPHAEROPSIS OLIVACEA CAUSING BROWN SPINE ROT ON ALHAGI MAURORUM IN IRAN

Camelthorn (Alhagi maurorum) is an importunate weed in cultivated fields and fallow lands in Iran. A disease at high severity level (69%) was observed on A. maurorum grown in arid area in Kermanshah province in September 2015. The disease was characterized by irregular brown lesions on the spines and profuse black pycnidia on aerially plant tissues. A Microsphaeropsis sp. was isolated from the lesions. The fungus was grown on oat meal agar (OA) at 24 C for seven days. Colonies of single-spore isolates were greenish brown with white aerial hyphae, had semi-immersed pycnidia in agar. Microscopic examination revealed that conidia were solitary, initially hyaline, becoming pale brown, 1-2-guttulate, ellipsoidal to subcylindrical with obtuse ends, straight to slightly curved, 0(-1) septate, (5-)6-7(-10.5) × 4-4.5 μm. The rDNA ITS was sequenced (GenBank accession No. KX171366) and found identical (99%) to sequences of Microsphaeropsis olivacea isolates. Pathogenicity of the isolates was confirmed by inoculating the healthy pinpricked spines with spore suspension (106 conidia/ml) or spraying with sterile distilled water as a control. Typical symptoms were produced on the inoculated spines after ten days. The pathogen was re-isolated from the infected spines while the controls remained symptomless. M. olivacea has been previously reported from Pirus laricio, and Sarothamnus sp. in Europe (Chen et al., 2015). To our knowledge, this is the first record of M. olivacea as a causal agent of A. maurorum in Iran and worldwide, and may have a potential for biocontrol of the weed.

First Report of Stem Rot of Stevia Caused by Sclerotinia sclerotiorum in North Carolina.

Stevia (Stevia rebaundiana Bertoni) is an emerging perennial crop in the United States. The crop is grown for 3 to 5 years with two harvests per growing season. Stevia contains numerous glycosides that are used as a natural noncaloric sweetener, and in 2008 was approved by the USDA as a sugar substitute. In commercial plantings of second-year stevia in North Carolina, diseased plants were observed in April and May of 2013. Diseased plants were observed in several counties in the state in fields that had been planted primarily in a corn-soybean rotation prior to stevia planting. Symptoms included wilting, chlorotic leaves, necrotic leaves at the base of the stem, bleached stem lesions, and dead plants. Symptomatic plants often also had tufts of white hyphae present on stems and large, irregularly shaped 2- to 8-mm black sclerotia frequently were present on the base of the stem. Isolations from infected stem tissue were made on potato dextrose agar amended with 50 μg/ml of streptomycin sulfate and penicillin G. Based on hyphal and sclerotial characteristics, isolates were tentatively identified as Sclerotinia sclerotiorum (Lib.) de Bary (4). Koch's postulates were confirmed on 10-week-old Stevia plants cv. G3 grown in the greenhouse in 10-cm-diameter pots containing a sterile 1:1:1 sand, loam, media mix. Oat grains infested with one isolate obtained from diseased field plants served as the inoculum. Oats were sterilized on three consecutive days, inoculated with colonized agar plugs of S. sclerotiorum, and then incubated at room temperature until they were thoroughly colonized. Three infested oat grains were buried 1 cm deep approximately 2 cm from the base of the plant in each of the six test pots and plants were observed over a 3-week period for symptoms. Symptoms developed on all plants within 5 days of inoculation. Leaves began to wilt, then turned chlorotic and necrotic, with stem lesions and sclerotia present at the base of the plant. Isolations were taken from infected stem tissue and pure cultures were prepared for molecular identification. Uninoculated control plants did not develop symptoms. Pathogen identification was confirmed using universal primers ITS 4,5 and β-tubulin (2,3). Mycelium from the cultured greenhouse stem isolations were grown in potato dextrose broth. Mycelium samples were aspirated and lyophilized prior to DNA extraction. Extracted DNA was amplified through PCR with ITS and β-tubulin primers and sent for sequencing. Sequences were aligned using CLC Workbench. Sequences from ITS45 had 100% identity to S. sclerotiorum GenBank Accession No. KF859933.1, confirming S. sclerotiorum as the causal organism. The β-tubulin sequence was compared against the Broad Institute S. sclerotiorum whole genome shotgun sequence and was confirmed to have 100% identity to the beta tubulin chain (5). This is the first report of S. sclerotiorum on stevia in the United States. Chang et al. (2) reported a stem rot of stevia in Canada and confirmed S. sclerotiorum as the causal organism.

First Report of Pythium Root and Stalk Rot of Forage Corn Caused by Pythium arrhenomanes in Japan.

Corn (Zea mays L.) is the most important forage crop in Japan. It was cultivated on 92,000 ha in 2011 and was mainly used as whole crop silage for cattle feed. In September 2009, a root and stalk rot disease was detected on corn plants cultivated in Tochigi, located in the central region of Japan. The symptoms of the disease included wilting of whole plants after the R5 (dent) stage (2) with drooping ears. Roots turned black and their number decreased. Further, the stalks became hollow and soft and harbored white hyphae. This tissue deterioration made machine harvest difficult. We obtained seven isolates of a Pythium-like organism by single hypha isolation from surface-sterilized pieces of diseased roots and stems on water agar and deposited one of the isolates at the NIAS genebank, Japan, under the accession no. MAFF511547. The isolate was grown in the dark on V8 juice agar medium for 10 days to produce oogonia. The oogonia were globose, light brown to yellow, smooth, 23.9 to 30.5 μm in size, and had 1 to 8 antheridia. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick. The morphology of the isolates was similar to that of Pythium arrhenomanes Drechsler and consistent with the species description (3). We analyzed the rDNA-ITS region sequences of the isolate as described by Kageyama et al. (1). The sequence (GenBank Accession No. AB903904) showed 99.1% (783/790 bp) similarity with that of P. arrhenomanes (AY598628). On the basis of morphological and rDNA sequence similarities, we identified the isolates obtained from corn as P. arrhenomanes. The pathogenicity of the isolate was confirmed by planting corn seedlings of the commercial Pioneer Brand hybrid 36B08 immediately after germination in five replicate pots containing soil mixed with 5% boiled barley grain by weight, incubated with or without the isolate for 7 days. After 10 days of incubation in a greenhouse at 20 to 25°C, only the inoculated plants exhibited symptoms of root and stalk rot. Since the inoculated organism was readily re-isolated from the diseased stems and roots, the pathogenicity of the isolate was confirmed. For field observation, the same hybrid of forage corn was sown in the fields in Nasushiobara, Tochigi, on 16 May 2011. The hybrid was sown in a row of 2 m, with 20 seeds planted at a distance of 10 cm with two replicates. For inoculum, the isolate was cultured on 5-cm-long wooden toothpicks, previously soaked in potato dextrose broth and placed on a V8 agar plate for 7 days at 25°C in the dark until covered by hyphae. The toothpicks were pierced into wounds made on the stems of corn plants, approximately 10 cm above the ground, using a thin iron needle. The wounds were about 2 mm in diameter and 2 cm deep. Field inoculation was conducted in late July at the R1 (silking) growth stage. Disease symptoms were observed in mid-September at R5, and only those plants that were inoculated with the toothpicks harboring the hyphae exhibited the typical stem rot symptoms. To our knowledge, this is the first report of root and stalk rot caused by P. arrhenomanes in forage corn in Japan.

First Report of Stem and Root Rot of Stevia Caused by Sclerotium rolfsii in North Carolina.

Stevia (Stevia rebaundia) is an emerging crop in the United States. Once established, the crop is grown for 3 to 5 years and is typically harvested twice per growing season. Stevia leaves contain multiple glycosides that are used as a natural noncaloric sweetener that was approved by the USDA in 2008 as a sugar substitute. In commercial plantings of Stevia in North Carolina, wilting and death of plants in first- and second-year plantings were observed in 2012 and 2013. Diseased plants were observed in multiple counties in the state, with first symptoms observed in May of each year and continuing through the summer months. Prior to Stevia, these fields had been planted primarily in a corn-soybean rotation. Symptoms began as moderate to severe wilting of young shoots and chlorosis of leaves, rapidly followed by death of stems and rotting of roots. White mycelial growth was frequently observed at the base of stem tissue. Theses characteristic hyphae of Sclerotium rolfsii were often accompanied by the presence of abundant white to brown sclerotia. Isolations from infected root and stem tissue were made on potato dextrose agar amended with 50 μg/ml of streptomycin sulfate and penicillin G. Isolations from diseased tissue yielded characteristic white hyphae of S. rolfsii (1,3). Numerous sclerotia 0.5 to 2 mm in diameter developed following 4 to 7 days of mycelial growth. Sclerotia were initially white and melanized turning brown with age. To verify pathogenicity, 10-week-old Stevia seedlings were transplanted in 10-cm diameter pots containing sterile 1:1:1 sand, loam, media mix. Inoculum consisted of oat grains infested with one isolate obtained from the field plants. Oats were sterilized on three consecutive days and then inoculated with colonized agar plugs of S. rolfsii. Oats were incubated at room temperature to allow the fungus to thoroughly colonize the oats. Three infested oat grains were added to each test pot and plants were then observed over a 3-week period. Symptoms were observed within 5 days on most plants and included chlorotic leaves, bleached stems, wilting, and necrotic roots. White mycelium and abundant sclerotia were found at the base of plants. Uninoculated plants did not develop any symptoms. This is the first report of S. rolfsii on Stevia in the United States. Kamalakannan et al. (2) reported a root rot disease of Stevia in India and confirmed S. rolfsii as the causal agent.

Monascus azaphilone pigment biosynthesis employs a dedicated fatty acid synthase for short chain fatty acyl moieties

The biosynthetic gene cluster of Monascus azaphilone pigments (MAzPs) encodes a canonical fatty acid synthase, MpFAS2. It is thus proposed that MpFAS2 plays a role in MAzP biosynthesis by supplying short chain (C8 and C10) fatty acyl moieties. Targeted gene inactivation of MpfasB2 in Monascus purpureus generated an F9 mutant, which developed white hyphae that discharged a yellow color on potato dextrose agar. High-performance liquid chromatography analysis demonstrated that F9 was incapable of producing MAzP and accumulated a wide array of chromophoric compounds instead. The main compound found in F9 was monascusone A, a hydrogenated azaphilone lacking a fatty acyl moiety. Gas chromatography analysis of the fatty acid methyl esters indicated that there was no significant difference in the cellular fatty acyl (C16 and C18) contents between WT and F9. The present study demonstrates that the dedicated fatty acid synthase is required to decorate the azaphilone polyketides in MAzP biosynthesis.

First Record of Acremonium Wilt in Tomato from Pakistan.

During a survey in May 2011, tomato (Lycopersicon esculentum Mill.) plants were observed exhibiting wilt symptoms on the vegetable farm of the University of the Punjab Lahore (31°32'59″ N, 74°20'37″ E), Pakistan. Plants were pale yellow and stunted. Diseased plants were scattered in the field either singly or in groups of two or three. Lower leaves turned yellow following necrosis and shedding from the plants. Roots of diseased plants were dark brown in comparison to the healthy ones. Vascular browning was observed when infected stems were split longitudinally and sectioned transversely. Necrotic vascular strands were also observed in infected stems. With a stereoscope, white hyphae were noticed in the vascular region of infected plants. Stained cross sections of stems of wilted plants showed fungal hypha. A diseased plant sample was taken to the First Fungal Culture Bank of Pakistan (FCBP) for identification of the pathogen. Conidia appeared on potato dextrose agar after incubation in diurnal light, and were unicellular, smooth, thin-walled, and oval in shape (3 to 6 × 1 to 3 μm), forming aggregations on the tip of conidiophores. With the help of diagnostic keys and available literature, the isolated fungus was identified as Acremonium strictum W. Gams. The fungus formed whitish flat colonies with deposition of gum granules on its surface. Hyphae and phialides were hyaline and non-pigmented. Phialides with expanded bottoms were elongated measuring 25 to 40 μm and solitary in position at right angles to the filament. To further confirm identification, amplification of the ITS1 and ITS2 regions was performed with universal primers ITS1 and ITS4 (4). The ITS sequence obtained was submitted to NCBI as accession JQ916901. The culture was deposited in FCBP (1099), Institute of Agricultural Sciences, University of the Punjab, Pakistan. A pathogenicity test of the isolated fungus was performed by inoculating 15 healthy tomato plants cv. Rio Grande in three replicates. The spore suspension (2 × 105 conidia/ml of sterile distilled water) was prepared from 12-day-old cultures of the fungus grown from a single spore on PDA in continuous light. Control plants were treated with sterile distilled water. Tomato plants inoculated with the fungus started showing wilting symptoms within 2 weeks, while the control remained healthy. A. strictum was re-isolated from inoculated wilted plants, thus satisfying Koch's postulates. Previously, A. strictum was a reported cause of wilt in many plants including Chrysanthemum maximum (2), watermelon (1), and Gladiolous grandiflorus (3). To our knowledge, wilt in tomatoes has been reported only from Fusarium oxysporum and Pseudomonas solanacearum from Pakistan. To our knowledge, this is the first record of Acremonium wilt in this economically important crop.

First Report of Phomopsis Seed Decay of Soybean Caused by Phomopsis longicolla in South China

Phomopsis seed decay of soybean (Glycine max (L.) Merr.) causes poor seed quality and suppresses yield in most soybean-growing areas in the world. The disease is caused primarily by Phomopsis longicolla Hobbs. During the spring of 2010, soybean seeds without symptoms were planted in the fields but emergence was poor and the emergence rates ranged from 30 to 70% in south China. Approximately 3,000 symptomless seeds were randomly collected from seven fields at three locations in Guangzhou, Nanchang, and Wuhan. Seeds were surface disinfected with 1% sodium hypochlorite for 12 min, rinsed in sterile distilled water three to four times, and placed on 2% agar. Plates were then incubated at 26°C under 16/8-h photoperiod for 3 to 4 days. About 10 to 20% of the seeds produced white hyphae that spread rapidly and covered the whole seed. The hyphae from fungal isolates were transferred to potato dextrose agar (PDA) and incubated at 26°C in the dark. After 3 to 4 weeks, conidia were elliptical with two oil drops at both ends and hyaline (6.2 to 7.2 × 2.6 to 3.2 μm). The cultural and morphological characteristics of the isolates corresponded with the description of P. longicolla (2). Colonies on PDA were floccose, dense, and white. Stromata were large, black, and spreading. To confirm the morphological identification, the ribosomal internal transcribed spacers (ITS1-5.8S-ITS2) from three isolates were sequenced (GenBank Accession Nos. JQ899030, JQ899031, and JQ899032). BLAST analysis indicated that the isolates had 99% nucleotide sequence identity with P. longicolla (GenBank Accession Nos. AY857868.1, EF026104, and HQ130441.1). Pathogenicity tests were conducted on 2-week-old soybean seedlings (3). A mycelial plug (3 mm in diameter) from the margin of 1-week-old PDA culture of the Wuhan isolate was individually placed mycelial side down directly on the top of cut stem 1 to 2 cm above cotyledon node of the soybean seedling. PDA plugs without the fungus was used as the negative control. All seedlings were kept in a growth chamber at 26°C with 92 to 94% relative humidity. After 2 weeks, all inoculated seedlings showed browning, stem wilt, and the lesions were 0.3 to 2.0 cm long. No symptoms were observed in the control plants. P. longicolla was reisolated from the infected seedlings. The pathogenicity test was repeated three times. Soybean stem blight caused by P. longicolla has been reported in northeast China (1). To our knowledge, this is the first report of P. longicolla causing Phomopsis seed decay of soybean in south China. This report will establish a foundation for developing a program for screening germplasm for resistance to this disease in south China.

Sclerotinia Wilt of Hop (Humulus lupulus) Caused by Sclerotinia sclerotiorum in the Pacific Northwest United States.

In June 2009, wilted hop bines were observed in a yard in Marion County, OR. The wilt was associated with a stem rot that occurred ~1 m from the ground near the point where bines are tied together for horticultural purposes. Samples of affected stems were submitted to the Oregon State University Plant Clinic. White hyphae and large, black sclerotia were present on the stems, with a clear delineation between healthy and diseased tissue. The pathogen was identified as Sclerotinia sclerotiorum based on morphological characters. In June 2011, bine wilting was observed on the same farm but in a different hop yard (cv. Nugget) ~10 km from the 2009 occurrence. Affected plants had upward curled leaves with necrotic margins or wilted bines that were severed at the soil line. Wilted bines tended to have smaller diameters than bines with foliar symptoms only. Of 100 plants examined, 75% displayed some foliar symptoms and 66% had at least one bine that was wilted. Yield loss was estimated at 10 to 20% due to bine wilting before cone development. Unlike the 2009 occurrence, wilted bines did not display aerial signs of S. sclerotiorum. Rather, water-soaked lesions covered in white, cottony mycelium were apparent on affected stems 2.5 to 5 cm below the soil surface, some bearing large, irregularly shaped sclerotia. Isolations made onto potato dextrose agar yielded isolates with rapid growth rates and morphological characters consistent with S. sclerotiorum (1). DNA was extracted (2) and pathogen identity was confirmed by PCR amplification and sequencing of the internal transcribed spacer regions from isolates SS001 and SS002 as described before (4). The amplicons were sequenced bidirectionally and consensus sequences were 100% similar to S. sclerotiorum (GenBank No. AAGT01000678.1). Two nucleotide polymorphisms were present that differentiated the sequences from those of 12 S. trifoliorum accessions in GenBank that could be aligned (2). Greenhouse assays utilizing a toothpick inoculation procedure (3) were conducted to fulfill Koch's postulates. Stems of five 4-week-old hop plants of cv. Agate were pierced with a toothpick colonized with S. sclerotiorum. Five control plants were similarly inoculated with toothpicks without the fungus. Inoculated plants developed symptoms similar to those observed in the field within 11 days; four of five plants inoculated with isolate SS001 and two of five plants inoculated with isolate SS002 completely wilted. S. sclerotiorum was reisolated from all inoculated plants but not the control plants. To our knowledge, this is the first report of Sclerotinia wilt on hop in Oregon or the Pacific Northwest (1), where nearly all commercial hop production occurs in the United States. The disease appears to be localized to a limited number of yards, although given the widespread distribution and host range of S. sclerotiorum, it is plausible that the disease may occur in other yards. Recurrent outbreaks and spread of the disease among yards on the affected farm suggests that Sclerotinia wilt has the potential to become a perennial problem on hop and efforts to limit the introduction of S. sclerotiorum into other yards are warranted.

Revision of Glomeromycetes with entrophosporoid and glomoid spore formation with three new genera

New ribosomal gene analyses reveal that Entrophospora is non-monophyletic and its type species E. infrequens closely related to Claroideoglomus species, which supports transfer of the Entrophosporaceae from Diversisporales to Glomerales as well as the 'ancestral' Claroideoglomus spp. to Albahypha gen. nov. Entrophospora baltica, supported as a separate clade within Diversisporales, is designated as type species for the new monospecific Sacculosporaceae. Entrophospora nevadensis, phylogenetically close to Diversipora spp. and Otospora bareae, is transferred to Tricispora gen. nov. (Diversiporaceae). Entrophospora, Sacculospora, and Tricispora are morphologically distinguished by spore wall structure, pattern of the two spore pore closures proximal and distal to the sporiferous saccule, and relative spore and sporiferous saccule sizes. The shape of the white hyphae subtending the spore base separates Albahypha spp. from Claroideoglomus spp.

Molecular phylogenetics of porcini mushrooms ( Boletus section Boletus)

Porcini ( Boletus section Boletus: Boletaceae: Boletineae: Boletales) are a conspicuous group of wild, edible mushrooms characterized by fleshy fruiting bodies with a poroid hymenophore that is “stuffed” with white hyphae when young. Their reported distribution is with ectomycorrhizal plants throughout the Northern Hemisphere. Little progress has been made on the systematics of this group using modern molecular phylogenetic tools because sampling has been limited primarily to European species and the genes employed were insufficient to resolve the phylogeny. We examined the evolutionary history of porcini by using a global geographic sampling of most known species, new discoveries from little explored areas, and multiple genes. We used 78 sequences from the fast-evolving nuclear internal transcribed spacers and are able to recognize 18 reciprocally monophyletic species. To address whether or not porcini form a monophyletic group, we compiled a broadly sampled dataset of 41 taxa, including other members of the Boletineae, and used separate and combined phylogenetic analysis of sequences from the nuclear large subunit ribosomal DNA, the largest subunit of RNA polymerase II, and the mitochondrial ATPase subunit six gene. Contrary to previous studies, our separate and combined phylogenetic analyses support the monophyly of porcini. We also report the discovery of two taxa that expand the known distribution of porcini to Australia and Thailand and have ancient phylogenetic connections to the rest of the group. A relaxed molecular clock analysis with these new taxa dates the origin of porcini to between 42 and 54 million years ago, coinciding with the initial diversification of angiosperms, during the Eocene epoch when the climate was warm and humid. These results reveal an unexpected diversity, distribution, and ancient origin of a group of commercially valuable mushrooms that may provide an economic incentive for conservation and support the hypothesis of a tropical origin of the ectomycorrhizal symbiosis.

KARAKTERISTIK AKAR BEREKTOMIKORIZA PADA Shorea pinanga, Pinus merkusii DAN Gnetum gnemon

Morphology and anatomy characteristics often used to identify ectomycorrhizal fungi. We used three Scleroderma spp. (Scleroderma columnare, S. dictyosporum), and S. sinnamariense) and inoculated to Shorea pinanga, Pinus merkusii, and Gnetum gnemon. After 6,8, and 10 months, each root tips were collected to determined hyphae colour, branching pattern, clamp-connection, hartig net and mantle. This result revealed that S. sinnamariense did not form association with S. pinanga and P. merkusii but form association with G. gnemon. On the other hand, S. columnare and S. dictyosporum could form association with all the host plants. S. columnare and S. dictyosporum formed white hyphae while S. sinnamariense formed yellow hyphae with monopodial branching pattern. The depth of hartig net and mantle was increased by timed. Key words: ectomycorrhizal fungi, hartig net, mantle, Scleroderma

Outbreak of Powdery Mildew Caused by Erysiphe cruciferarum on Spider Flower (Cleome hassleriana) in Italy

Spider flower (Cleome hassleriana L. [synonym = C. spinosa]) is used as a tall border in parks and gardens worldwide. During July 2008, severe outbreaks of a powdery mildew were observed in a public park in Torino (northern Italy). Leaves were covered with dense, white hyphae and conidia, especially on the adaxial surface. Hyphae were also present on petioles and fruits, but not on petals and stems. As the disease progressed, infected leaves turned chlorotic, curled, and wilted. Conidia were hyaline, cylindrical, single, and measured 31.1 to 48.2 × 12.9 to 17.6 μm (average 37.1 × 15.6 μm). Germ tubes terminating in a moderately lobed appressorium were produced terminally. The cylindrical foot cells of the erect condiophores were 19.2 to 27.8 × 6.5 to 8.6 μm (average 23.3 × 7.7 μm). Fibrosin bodies were absent. Chasmothecia were observed mostly on the lower surfaces of leaves. At maturity, they were dark amber and spherical with a diameter of 92.9 to 151.0 μm (average 121.4 μm). Each chasmothecium contained six stalked asci (average size 63.7 × 35.9 μm). Each ascus contained four ellipsoid ascospores that measured 17.3 to 26.4 × 10.9 to 15.6 μm (average 23.3 × 12.8 μm). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced (1). The 602-bp sequence was deposited in GenBank under the Accession No. GQ149478 and was 99% similar to that of Erysiphe cruciferarum (Accession No. EU140958). As proof of pathogenicity, diseased leaves of C. hassleriana were pressed against leaves of three healthy 4-month-old potted plants of the same species for 10 min. Three noninoculated plants served as controls. Inoculated and noninoculated plants were maintained in a greenhouse at 22 to 25°C in isolation. After 11 days, typical powdery mildew colonies developed on inoculated plants. Noninoculated plants did not develop symptoms. The pathogenicity test was repeated once. Powdery mildew on C. hassleriana caused by E. cruciferarum was reported in Italy (2) but the pathogen was not characterized. Herbarium specimens are deposited at AGROINNOVA Collection, University of Torino, Italy.

First Report of Rhizoctonia solani Subgroup AG 1-ID Causing Leaf Blight on Durian in Vietnam.

During the rainy season in Vietnam, leaf blight disease caused by Rhizoctonia solani often occurs on 3- to 5-year-old durian (Durio zibethinus). Symptoms appear as large, pale brown, blighted lesions with an irregular border. In excessive moisture conditions, yellowish white hyphae appear on the lesions, and the affected leaves turn dark brown and wilt. There are no reports describing the anastomosis groups (AG) and subgroups of Rhizoctonia solani occurring in durian. In June of 2004, two isolates of R. solani were obtained from leaf blight lesions on durian growing in Binh Duong and Dong Nai provinces. The durian isolates were identified as AG 1 based on hyphal anastomosis. In pathogenicity tests, the durian isolates infected cucumber, mung bean, and leaf mustard seedlings grown on water agar in petri dishes. The rDNA-ITS sequence of the durian isolates was determined (GenBank Accession Nos. EF197797 and EF197798) and aligned with those of AG 1-IA, AG 1-IB, AG 1-IC, and AG 1-ID available in the GenBank database. The sequence similarity of the total rDNA-ITS region (including 5.8S) within the durian isolates was 99.9%. The sequence similarity of the durian isolates and AG 1-ID isolates was 99.1 to 100%, but similarity with other AG 1 subgroups was 89.1 to 94.0%. The results suggest that the two Vietnam durian isolates of R. solani are members of AG 1-ID. AG 1-ID has only been reported causing necrotic leaf spots on coffees in the Philippines (1). To our knowledge, this is the first report of R. solani AG 1-ID on durian and the first report of the presence of R. solani AG 1-ID in Vietnam. Reference: (1) A. Priyatmojo et al. Phytopathology 91:1054, 2001.

First Report of Rhizome Blight of Ginger Caused by Binucleate Rhizoctonia AG-R in China.

In 2004, rhizome blight of ginger (Zingiber officinale (Willd.) Roscoe) (cv. Yunnanxiaojiang) occurred in the Kunming District of China. The surface of ginger rhizome after harvest was crimpled and covered with white hyphae. Initial symptoms on ginger were wilting on the stem and the color of the rhizome turned from white to light brown with no lesion formation. After 2 weeks, the surface of ginger rhizome was covered with white hyphae and a dry rot set in under humid conditions. The yield loss in ginger almost reached 50% because of the disease. An AG-R tester isolate paired with the unknown 37 isolates of Rhizoctonia spp. from the diseased ginger rhizomes caused a C2 reaction that confirmed their identity. Isolates of AG-R (GenBank Accession Nos. DQ885780 and DQ885781) had 100% sequence similarity with 5.8S rDNA-ITS with the AG-R tester isolate (GenBank Accession No. AF354082). To produce infected soil inoculum, 10 isolates were cultured on potato dextrose agar in a 9-cm petri dish for 3 to 4 days and then covered with approximately 20 g of autoclaved soil and kept at 25°C for 3 to 4 days. Seedlings of ginger (cv, Yunanxiaojiang) were planted in natural potting soil at a density of one plant per vinyl pot (8 cm in diameter, 9 cm high) and grown in the greenhouse for 7 days. Each seedling was inoculated with 7 g of infested soil by placing it around the rhizome. Control plants were inoculated with autoclaved soil. The experiments were carried out three times, each time with three replicates in a growth chamber kept at 25 and 16°C with a 16-h light and 8-h dark photoperiod. After 14 days, the disease severity was recorded based on a scale in which - = no symptoms; + = small lesions on seedlings, no blight; ++ = seedling blight; and +++ = plant dead. All of the 10 tested AG-R isolates caused ginger seedling blight. Rhizoctonia spp. was reisolated from these plants, confirming its pathogenicity. To our knowledge, this is the first report of rhizome blight of ginger caused by Rhizoctonia spp. and binucleate Rhizoctonia AG-R in China.