Universal Primers ITS4 Research Articles

The Formation of Steppe and Protective Afforestation Basement: Theoretical and Applied Aspects

Introduction. Fusarim are widely spread in forest nurseries. It is difficult to identify Fusarium species using microbiological methods. The goal of this study was to identify the micromycetes of Fusarium genus isolated from Pinus sylvestris and Picea abies seedlings and to develop a method of species identification for F. oxysporum, F. verticilliodes, and F. graminearum based on the restriction analysis of amplified ribosomal DNA. Objects and research methodology. The universal primers ITS1 and ITS4 were used in the study. A PCR-product of about 550 base pairs for each Fusarium species was generated. Three Fusarium species (F. oxysporum, F. verticilliodes, and F. graminearum) were identified by the molecular genetic analysis based on the sequence of ITS rDNA region. Since these fungi are difficult to distinguish by morphological markers, a simple method based on the digestion by restriction endonucleases of ITS1-5,8S-ITS2-28S region was developed. Five restriction endonucleases (AluI, BstMBI, BstHHI, BsuRI, and HpaII) were used for digestion of the studied DNA. Research results. Only HpaII and BstHHI allowed to get the unique RFLP banding patterns for each Fusarium species. When using the restrictase HpaII, a unique fragment over 440 bp for F. oxysporum and two fragments over 300 bp and 160 bp for F. verticilloides were identificated. The digestion of PCR fragments by using BstHHI enzyme revealed two specific fragments (300 bp and 280 bp) for F. oxysporum and two fragments (300 bp and 190 bp) for F. verticilloides. Conclusion. The PCR-RFLP analysis of the amplified ITS region was proved to be a useful molecular tool for identification of F. oxysporum, F. verticilliodes, and F. graminearum.

First Report of Tilletiopsis pallescens Causing White Haze on Apple in Croatia

During 2014, apples ‘Pink Lady®’, showing white haze (WH) symptoms, were harvested in an orchard near Vratisinec (Croatia). Fungi were isolates from symptomatic epidermal tissue on Rose Bengal Chloramphenicol agar. After 7 days of incubation in the dark at 26°C, from whitish to cream colonies with irregular margin, single spore were isolated and transferred on Potato dextrose agar (PDA). Pathogenicity was tested on 20 apples ‘Pink Lady®’ and the pathogen was inoculated by aerosol diffusion on the sterilized surface. Symptoms occurred and T. pallescens was reisolated from inoculated fruit on PDA. Control fruits were symptomless. After 7 days at 26°C, the colonies and the morphology of conidia were the same as those of the original isolates. PCR amplification was carried out, from single spore DNA extraction, using universal primers ITS1 and ITS4. Two amplified sequences (AN KR269863 and KR269864) were blasted in Genbank obtaining 100% homology with strains of Tilletipsis pallescens. To confirm the species DNA sequences were aligned with CLUSTAL W with closely related species of Tilletiopsis (T. pallescens GQ281316.1, DQ317636.1 ; T. washingtoniensis DQ025483.1 ; T. lilacina AB025683.1, AB025689.1T ; T. cremea AB025690.1 ; and an undefined species Tilletiopsis sp. GQ281313.1) and a phylogenetic analysis with the Neighbor Joining method based on Maximum Composite Likelihood model (bootstrap 1, 000) was performed (supplementary material). The phylogenetic tree confirmed the identity of the isolates to the species Tilletiopsis pallescens. To our knowledge, this is the first report of Tilletiopsis pallescens causing postharvest white haze on apple in Croatia.

Direct colony polymerase chain reaction for rapid identification of yeasts isolated from blood specimen

Context: Bloodstream infections (BSIs) caused by yeasts have an increasing frequency due to the growing population of immunosuppressed individuals. Among yeasts, Candida remains the most prevalent species with the increase in the incidence of non-albicans Candida species. Apart from Candida, other yeasts are also involved in causing BSI. High mortality associated with Candida and other yeast infection can be reduced by prompt and appropriate antifungal therapy. Hence, rapid identification and speciation of yeasts isolated from blood play a significant role in the management of the patients. Since conventional methods used for speciation of Candida and other yeasts are laborious, time-consuming and often unclear, rapid and accurate molecular techniques are required. Materials and Methods: Instead of using purified genomic DNA as template for polymerase chain reaction (PCR), we used yeast colony and cell suspensions in water and 0.10M potassium hydroxide as template for PCR. Candida albicans, Trichosporon and Cryptococcus neoformans were used as reference strains. Further, a total of 100 yeast isolates were also tested. All reactions were performed using the universal fungal primers ITS1 and ITS4; the PCR products were then digested with restriction enzyme (Msp1). Results: Direct colony PCR (DCPCR) produced sharp and distinct bands compared to the cell suspensions with the reference strains. All the 100 clinical isolates tested also produced distinct bands. Conclusion: DCPCR approach not only reduces the DNA template preparation time but is also easy, rapid and reduces the cost of PCR.

Molecular Taxonomy Of Galactomyces Spp. And Dipodascus Capitatus Associate With Dairy Based On Rdna Sequence Analysis In Iraq

Galactomyces spp is a telomorph of Geotrichum candidum (anamorph name). is used as a culture for cheese making and in some traditional fermented milks, few studies have assessed the genetic diversity of Galactomyces spp that exist in traditional cheese making facilities. The aim of this study isolation and molecular taxonomically treated of Galactomyces spp and other Candida spp. correlated with dairy products. The results showed that most dairy samples companioned with two species of Galactomyces: G.candidum and G.geotrichum , D. capitatus and four species of Candida spp.: Candida krusei , C. kefyr, C. utilis and C.glabrata .A total of 23 fungal isolates were diagnosed based on universal primers ITS1 / ITS4 to amplify the Internal transcribed region spacer. Four doubtful G.candidum isolates showed unique sizes of products PCR ranged between 380-400 bp. other Candida PCR product ranged 420-780bp. Sequence analysis identified a doubtful G.candidum into G. candidum and G.geotrichum with 97% similarities and with D. capitatus showed 93% similarities, few intraspecific variation were observed at 312-380bp in the amplicons from the primer pair ITS1-ITS4 commonly are from 380 to 400 bp for G. candidum , G.geotrichum and D. capitatus and other variation in the ITS region of Candida spp under our interest .

New Report of a Sweet Basil Leaf Blight Caused by Cochliobolus lunatus in India.

Sweet basil (Ocimum basilicum), a member of the Lamiaceae, is used as an ornamental as well as a culinary herb. It is a rich source of the phenolic compound methyl chavicol and is used as a traditional medicinal plant in India, where the crop is grown on ~2,500 ha annually (4). The species is native to India, where it has been cultivated for >5,000 years. During the rainy season, August of 2013, a severe leaf blight was observed on 30- to 45-day-old sweet basil plants in experimental fields (approximately 5 ha) at the CSIR-CIMAP and adjoining areas in Lucknow. Initial symptoms comprised small, irregular, necrotic lesions that coalesced into a leaf blight. Infected parts of the leaves turned black during wet and humid conditions. The incidence of symptoms ranged from 20 to 30%. Infected leaf samples were cut into small pieces and surface-sterilized with 1% sodium hypochlorite for 1 min, followed by two rinses in sterilized, distilled water. The leaf pieces were then blotted dry with sterilized filter paper, placed onto potato dextrose agar (PDA), and incubated at 28°C for 3 to 5 days. Blackish-brown fungal colonies developed. Microscopic examination revealed the presence of brown conidiophores that were cylindrical, septate, unbranched, and straight or geniculate near the apex. Conidia were three-septate, mostly curved at the third cell from the base, which was usually larger and darker than the other cells; intermediate cells were brown or dark brown; terminal cells were subhyaline or pale brown and 16 to 23.5 × 8.5 to 11.5 μm (the average size of 100 conidia was 19.9 × 10.18 μm). On the basis of these characteristics, the fungus was identified as Cochliobolus lunatus (anamorph Curvularia lunata (Wakk.) Boedijin) (1,2). The identification was confirmed by sequencing the internal spacer (ITS) region of ribosomal DNA (rDNA). Genomic DNA was extracted from five fungal isolates, using the 5 Prime Archive Pure DNA Cell/Tissue kit, and subjected to a polymerase chain reaction (PCR) assay with the universal primers ITS1 and ITS4 (5). The amplified product was cloned and sequenced. An NCBI-BLASTn search showed greatest homology (98% similarity) with the ITS sequence of C. lunatus (GenBank Accession No. DQ836800). The sequence was deposited in Genbank (KM272001). A pathogenicity test was carried out using 10, 30-day-old sweet basil (cv. CIM Soumya) plants in pots, by spraying a spore suspension (105 spores/ml) onto the leaves of each plant. Five plants treated similarly with sterilized, distilled water served as a control treatment. The plants were kept at 27 ± 2°C and 85 ± 3% RH for 8 to 10 days. Small, irregular, necrotic lesions appeared after 4 days on all inoculated leaves, while leaves of control plants remained asymptomatic. Fungi re-isolated from inoculated leaves resembled C. lunatus on the basis of microscopic and sequence data, fulfilling Koch's postulates. The fungus was not re-isolated from the control plants. C. guatemalensis has been reported to cause a leaf spot on sweet basil in Korea (3). To our knowledge, this is the first report of a sweet basil leaf blight caused by C. lunatus in India. C. lunatus has the potential to reduce the yield of sweet basil.

First Report of Aspergillus niger Causing Root Rot of Peanut in China.

Peanut (Arachis hypogaea) is one of the most important oil crops and food legumes worldwide. China sows approximately 3.5 million hectares each year and produces 40% of the world's peanuts. Fungal diseases are among the main biotic stresses affecting peanut production. Root rot is a serious disease caused by several fungi. Pythium spp., Fusarium spp., and Rhizopus spp. are some of the root rot fungi that have been reported in China. In 2012 and 2013, root rot symptoms were observed in several fields in Laixi District, Qingdao City, Shandong Province, China. The first symptoms appeared in July. Initial symptoms of the disease were brown spots on the stem base and root. Affected plants were stunted, with leaf chlorosis, reduced growth, or sudden wilting. As disease progressed, the infected tissues showed brown discoloration and rot, and abundant dark brown and black powdery spores were visible on the surfaces of affected parts. Eventually, affected plants collapsed and died. To isolate the causal organism, roots and stems were cut into sections, which were surface-disinfected with 70% ethanol solution (v/v) for 20 s, soaked in 0.1% mercuric chloride solution for 50 s, rinsed with sterilized water three times, dried, placed on Czapek's Dox agar supplemented with chloramphenicol (100 μg/ml), and incubated at 28°C for 7 days. Fungal colonies were white initially and then covered with a dense layer of dark brown or black conidial heads. The conidial head was radiate; vesicles were nearly spherical and covered with irregular metulae and phialides. Conidia were globose or subglobose (3.0 to 5.5 μm in diameter), dark brown to black, with rough cell walls. Total genomic DNA was extracted from mycelia using the EasyPure Genomic DNA Kit (TransGEN, Beijing, China). The rDNA-ITS region was amplified using PCR with the universal fungal primers ITS1 and ITS4 (2). The purified products were separately sequenced in both directions using the same primer pair. The sequences (GenBank Accession No. KJ848716) obtained were 99% similar to the ITS sequence of isolates of Aspergillus niger. This, together with the morphological characters (1) described above, suggested that the microorganism we had isolated was A. niger. Koch's postulates were completed in the laboratory by inoculating peanut. Thirty Huayu20 peanut seeds were placed in a 500-ml sterile pot with 300 g of autoclaved soil. Twenty days after seedling emergence, 15 peanut plants were wounded with a needle and inoculated with 5 ml of conidia suspension (106 ml-1). The same number of peanuts were similarly wounded and inoculated with 5 ml of sterile distilled water to serve as controls in the same pot. All peanuts were kept in a randomized complete block design at 30°C under a 12-h photoperiod. After 7 days, disease symptoms similar to those observed in the field appeared in all inoculated but not in non-inoculated peanuts. The tests were repeated three times in the greenhouse. Koch's postulates were satisfied after re-isolating the A. niger from inoculated peanuts using the method described above. To our knowledge, this is the first report of A. niger causing root rot in peanut in China.

First Report of Phytophthora megasperma Causing Decline and Death on Celtis australis in Italy.

European hackberry (Celtis australis L.) is a popular shade tree mainly planted in southern Europe and known to be tolerant to dry and poor soils. In early autumn 2013, hackberry plants grown in soil in a commercial nursery located in the floodplain in Umbria region showed symptoms of wilting, dieback, and death. Up to 100% of the canopy was affected, and over 60% of the plants were symptomatic or dead. A Phytophthora species was consistently isolated from symptomatic 6-year-old plants by plating small pieces of collar and root tissues, cut from the margin of dark-brown necrotic lesions, onto P5ARPH selective medium (4). Pure cultures were obtained by single-hyphal transfers on potato dextrose agar (PDA). Sporangia, produced on pepper seeds in soil extract solution (3), were nonpapillate and noncaducous, measuring 34.0 to 85.0 × 22.0 to 50.0 μm. Oospores had an average diameter of 44 μm with mostly paragynous antheridia. On the basis of morphological features, the isolates were identified as P. megasperma Drech. (2). The identity was confirmed by sequencing the cytochrome c oxidase subunit II (Cox II) (5), which gave 100% identity with P. megasperma sequences available in GenBank (GU222070), and by sequencing the internal transcribed spacer (ITS) using the universal primers ITS4 and ITS6, which gave 99% identity with the AF266794 sequence from Cooke et al. (1). The sequences of one isolate (AB239) were deposited in the European Nucleotide Archive (ENA) with accession numbers HG973451 and HG973450 for Cox II and ITS, respectively. Pathogenicity tests were conducted in the greenhouse with isolate AB239 on eight 2-year-old potted European hackberry plants. Mycelial plugs (5 mm diameter) cut from the margins of actively growing 8-day-old cultures on PDA were inserted through the epidermis to the phloem at the collar level. Two plants were used as controls and treated as described above except that sterile PDA plugs replaced the inoculum. Inoculated plants were kept for 4 weeks in a greenhouse at 24 ± 2°C. During that period, inoculated plants showed wilting symptoms similar to those observed in the field. Lesions were evident at all the inoculation points progressing downward to the roots. Colonies of Phytophthora were isolated from the margins of lesions and identified as P. megasperma, thus fulfilling Koch's postulates. Controls remained symptomless. P. megasperma taxonomy is rather complex since it embraces different subgroups, including host specialized forms (formae speciales), some of which are recognized as biological species. Based on morphological and molecular data presented here, the Phytophthora isolates from hackberry belong to P. megasperma sensu stricto, which is included in the "pathogenic to a broad range of hosts" (BHR) group (1). This pathogen is rather polyphagous, attacking mainly fruit and ornamental woody plants, commonly Prunus spp., Malus spp., and Actinidia deliciosa. Like other homothallic Phytophthora species, it is particularly dangerous due to its abundant production of thick-walled resting oospores that enable long-term survival in the soil. To our knowledge this is the first report of P. megasperma sensu stricto (1) on C. australis and its family Ulmaceae/Cannabaceae.

First Report of Acrostalagmus luteo-albus Causing Red Rust of Needle Mushroom (Flammulina velutipes) in China.

Needle mushroom (Flammulina velutipes) is an edible and medicinal mushroom with annual production yields for China exceeding 2 million metric tons. Hebei province is one of the biggest producers of needle mushrooms in China. From 2009 to 2010, red rust-colored sporulation (fungal mycelia and conidia) was found on the substrate surface of white needle mushroom bags in more than 10 mushroom cultivation operations in Hebei. The rust-colored sporulation covered the substrate surface, where the development of the fruiting bodies was inhibited; the stipes were sparse and became light brown when the substrate was slightly affected. The fruiting bodies on severely affected substrate were unable to complete development or shriveled and died off on the substrate. More than 30 to 40% substrate bags were contaminated with the red rust mold, with approximately a 40% yield reduction in each production house. Single conidia were isolated from the red rust mold and cultured on potato dextrose agar at 25°C in the dark. The colony was round, compact, reddish-brown, and slow-growing (2 to 3 mm/d). Main conidiophore axes were erect, often branched in five to seven layers, tapered from the base to the upper part, pale reddish brown at the base, and almost hyaline at the apex. Two to five phialides usually verticillated at each level along the main stipe and its branches. Phialides were narrowly flask-shaped and only very slightly swollen at the base. Conidia were oval, measured 3.5 to 5.4 (4.5) × 2.3 to 3.6 (3.0) μm, L/W = 1.2 to 2.0 (1.5), and formed pale reddish brown, slimy heads. The isolate was tentatively identified as Acrostalagmus luteo-albus based on its morphological characteristics (2). For molecular analysis, the internal transcribed spacer (ITS) regions of ribosomal DNA from the isolate were PCR-amplified using universal primers ITS1 and ITS4. The resulting sequence (Accession No. KC127681) submitted to GenBank had a 99% identity to that of A. luteo-albus (JN545827) isolated from vineyard soil and Accession No. JQ387575 isolated from twig of declining persimmon tree. To confirm the pathogenicity of the isolate to needle mushroom, five bags of needle mushroom with stipes of 1 to 2 cm long were inoculated with a conidia suspension (105 conidia/ml) of the isolate and incubated at 13 to 15°C in the dark, while five non-inoculated bags (sprayed with sterile water only) were used as a control. Five days after inoculation, the development of the fruiting body was obviously inhibited, and a few growing fruiting bodies became light brown, compared with the non-inoculated fruiting bodies, which were growing healthily and fast and were white with no symptoms. The pathogen was re-isolated from the inoculated fruiting bodies. A. luteo-albus is primarily saprophytic but is sometimes pathogenic. Indeed, it has been associated with post-harvest rot of ginger rhizomes in Brazil (1). To our knowledge, this is the first report of A. luteo-albus causing damage to needle mushroom in China.

FIRST REPORT OF ROSEMARY LEAF SPOT CAUSED BY PHOMA GLOMERATA IN IRAN

Rosemary (Rosemarinus officinalis) is an ornamental and medicinal plant grown in Iran. During a survey in November 2012, symptoms of wilt and leaf spot were observed in almost half of rosemary fields of Kerman (southeast of Iran). Samples of infected leaves were surface sterilized with 0.5% sodium hypochlorite, rinsed with sterile distilled water, cultured onto potato dextrose agar (PDA) medium and incubated at 25°C for seven days. The isolated fungus produced a pale brown to dark green colony. Ovoid or ellipsoidal, hyaline, and aseptate conidia were produced abundantly in subglobose pycnidia. Numerous dictyochlamydospores and chlamydospores were also observed. Based on the morphological characters, the fungus was identified as Phoma glomerata (Boerema et al., 2004). To confirm the species of the fungus, DNA was extracted from a single spore isolate and the internal spacer regions (ITS) were amplified with universal primers ITS1 and ITS4. The resulting sequence (532 bp), which showed more than 99% identity with Phoma glomerata, was submitted to NCBI GenBank (Accession No. KM114267). To test the pathogenicity, two month old plants were sprayed by a suspension of 104 spores per ml, covered with plastic bags and incubated under greenhouse conditions at 25-28°C. Pale brown small spots were developed on an average of 31.48% of the leaves after seven days. This fungus has been previously reported from Iran on cucumber (Hatami et al., 2008) and Ficus elastica (Aghapour et al., 2009). To the best of our knowledge, this is the first report of Phoma glomerata on rosemary in Iran and possibly in the world.

FIRST REPORT OF Botrytis cinerea CAUSING POSTHARVEST GRAY MOLD OF TEJOCOTE (Crataegus mexicana) FRUIT IN MEXICO

In Mexico, tejocote (Crataegus spp.) is grown commercially on a total area of more than 900 ha. During November and December 2013, fruits of Crataegus mexicana var. chapeado showing a gray and firm rot were collected in commercial markets in Puebla. Small pieces of decayed fruits were surface-disinfected for 1 min in a 2% sodium hypochlorite solution, rinsed in sterile distilled water, and plated onto potato dextrose agar (PDA). A fungus was isolated whose colonies were initially whitish but turned gray with age. Black and irregular sclerotia were formed after 14 days of incubation at 20°C. Conidiophores were erect, subhyaline and dichotomously branched, and bore unicellular, ovoid-ellipsoid, subhyaline conidia measuring 7.7-12.7x6.1-9.5 μm. Based on morphology, the fungus was identified as Botrytis cinerea. Genomic DNA was extracted and the internal transcribed spacer (ITS) region of rDNA was amplified using the universal primers ITS5 and ITS4 (White et al., 1990). PCR products were purified and sequenced. The resulting sequence of 496 bp was deposited in GenBank (accession No. KM594622). BLAST search showed 100% identity with B. cinerea sequences KF010847 and KJ476697. To confirm the pathogenicity of the fungus, 10 tejocote fruits were surface-disinfected with 80% ethanol. A conidial suspension (1x106 spores ml-1) was sprayed on the surface of non-wounded fruits. Control fruits were sprayed with sterile distilled water. Typical gray mold symptoms with gray sporulating lesions were observed only on inoculated fruits after eight days. Koch s postulates were fulfilled when the pathogen was re-isolated from the diseased fruits. To our knowledge, this is the first report of B. cinerea causing postharvest fruit rot on tejocote in Mexico and worldwide.

First report of peppermint leaf spot caused by Alternaria alternata in Iran.

Peppermint (Mentha piperita), a herbaceous plant in the family Lamiaceae, is a medicinal herb widely grown in Iran. Symptoms of leaf spot were observed during a survey in peppermint fields of Kerman (southeast Iran) in November 2012. In some fields, more than 40% of the plants showed leaf spots. Samples of infected leaves were surface-sterilized with 0.5% sodium hypochlorite, rinsed with sterile distilled water, cultured onto potato dextrose agar (PDA) and incubated at 25°C. After seven days, dark olivaceous colonies developed which produced profuse golden brown, branched, and septate hyphae, and muriform conidia in long chains on straight and septate conidiophores. Based on the morphological characters, the fungus was identified as Alternaria alternata (Simmons, 2007). To confirm identification, DNA was extracted from a single spore isolate. The internal spacer (ITS) region was amplified using the universal primers ITS1 and ITS4 and sequenced. The resulting sequence (560 bp), which showed more than 99% identity with A. alternata isolates in BLASTn analysis, was submitted to GenBank (accession No. KM076936). Pathogenicity tests were performed by spraying suspensions of 104 spores per ml on the leaves of healthy peppermint plants. After 10 days, an average of 47.85% of the leaves of inoculated plants showed spots similar to those observed in the field. A. alternata has previously been reported as a leaf spot pathogen on peppermint from Poland (Zimowska, 2007) but, to our knowledge, this is its first report on peppermint in Iran.

First Report of Foxtail Millet Seedling Damping-Off Caused by Binucleate Rhizoctonia AG-A in China.

Foxtail millet (Setaria italica) is an important food and fodder grain crop in China. In June 2013, foxtail millet seedlings of cultivar Jigu19 showing symptoms of damping-off were observed with about 30% incidence in Handan, Hebei Province. Infected plants showed pale brown lesions on the stems and brown discoloration of the roots, which later turned into root rot. As a consequence, infected seedlings wilted and died prematurely. Segments of the diseased root tissue (5 mm long) were washed with sterile water, disinfected with 0.5% NaOCl for 2 min, rinsed with sterile water, placed on potato dextrose agar (PDA), and incubated at 25°C in the dark. Three isolates were obtained and designated HD-1, HD-3, and HD-5. Morphological characteristics of the three isolates were similar with white colonies bearing large amounts of floccose aerial hyphae and no production of sclerotia after 14 days. Hyphal cells were stained with DAPI and all the three isolates were binucleate. Total genomic DNA was extracted from the mycelial mat using CTAB (4). The rDNA internal transcribed spacer (ITS) region was amplified using the universal primer pair ITS1 and ITS4 (1). PCR amplicons were purified and sequenced. A BLASTn search revealed that the resulting sequences (GenBank Accession Nos. KM017960, KM017961, and KM017962 for HD-1, HD-3, and HD-5, respectively) shared 99% identity with other Ceratobasidium sp. AG-A isolates (JX913824, FJ440197). In addition, sequence identity of HD-1, HD-3, and HD-5 with each other was 99.8%, 99.7%, and 99.8%, respectively. Thus, the isolates were identified as Ceratobasidium sp. AG-A, i.e., binucleate Rhizoctonia (BNR) AG-A. Pathogenicity tests were conducted by placing autoclaved wheat seeds, each colonized with Rhizoctonia isolates at inoculum density of 25 propagules per gram (ppg), on the surface of a sterilized mix of soil, sand, and nutrient soil (1:1:1, v/v/v) in pots (3). Each pot contained five healthy seedlings of the foxtail millet cultivar Yugu 1, and every seedling was inoculated by placing three colonized wheat seeds under the sheath of foxtail millet at the 2- or 3-leaf stage; non-inoculated sterilized wheat seeds were used as a control. Plants were incubated at 25°C with 14 h light and 10 h dark in a growth room for 10 days, and then assessed for disease. Damping-off symptoms similar to those in the field appeared on inoculated plants; control plants were asymptomatic. BNR Rhizoctonia were re-isolated from diseased plants and confirmed to be AG-A based on morphological characteristics and rDNA-ITS sequence. BNR AG-A has been reported in China as a pathogen of Chinese mustard, Chinese cabbage, potato, and sugar beet, but there are no previous reports of its presence on foxtail millet. R. solani AG-1 and AG-4 are usually regarded as pathogenic to foxtail millet (2). To our knowledge, this is the first report of BNR AG-A causing foxtail millet damping-off in China. We found binucleate Rhizoctonia AG-A, the anamorph of Ceratobasidium sp., can infect foxtail millet at seeding stage, which may cause serious losses of the crop. Therefore, we should pay particular attention to it when considering seeding disease control and breeding disease resistance varieties of foxtail millet in the future.

Morphological, Pathogenic and Molecular Characterizations of Alternaria Species Causing Early Blight of Tomato in Northern India

Early blight disease samples were collected from six different states of India. Seventeen different isolates of Alternaria spp were obtained in pure culture which were designated as PB-2, PB-4, PB-13, PB-17, PB-19, HR-12, HR-44, HR-62, MP-6, MP-7, JT-4, JT-11, UP-7, UP-10, UP-15, UP-20 and RJ-T-1. Characterization based on culture colour and conidial dimensions (conidial length and breadth, and beak length) indicated that no distinguished variation of the isolates was observed on the colour parameters. Although, a wide variation was observed in the length of conidia and beak, no significant difference was observed in conidial breadth except RJ-T-1. Among the isolates, conidial length of five isolates viz., HR-12, UP-7, UP-10, UP-15 and RJ-T-1 ranged from 80 to 97 µm whereas others were of significantly lesser size (30–51 µm). Similarly, significant difference was observed in beak length. The PCR amplification of the fungal DNA using universal primers ITS1 and ITS4 and sequencing indicated that, out of 17 isolates, 12 (JT-4, JT-11, HR-62, HR-44, PB-2, PB-4, PB-13, PB-17, PB-19, UP-20, MP-6 and MP-7) were closely matching with A. alternata (JX993756) and five isolates (HR-12, UP-7, UP-10, UP-15 and RJ-T-1) with A. solani (JF796068). Further, pathogenicity test on tomato revealed that both A. alternata and A. solani isolates were of virulent category indicating that former is also an incitant of early blight in northern India.

First Report of Downy Mildew Caused by Hyaloperonospora camelinae on Camelina sativa in Slovenia.

Camelina or false flax (Camelina sativa), of the Brassicaceae, is an annual flowering plant native to Europe and Central Asia where it is grown commercially as an oilseed crop. At the end of May 2012, symptoms of downy mildew were observed on camelina plants grown in the Savinja Valley in Slovenia. The disease was found in four monitored fields (total area 3 ha), and the incidence ranged from 2 to 38% depending on the variety. Symptomatic plants showed whitish, abundant, and fluffy mycelia covering the stems, flowers, seed pods, and undersides of the leaves. The disease mainly affected the upper half of the plants, and the stems were reduced and distorted. During disease progression, the mycelium turned from gray to black. Microscopic observations revealed hyaline, straight conidiophores that were branched monopodially (3 to 4 times) with 6 to 12 re-curved tips/branch, and measured 140 to 300 × 12 to 20 μm. Conidia were hyaline, oval to broadly ellipsoidal, 24 to 29 × 18 to 24 μm. Oospores formed in necrotic stem and leaf tissues were dark brown and measured 30 to 38 μm in diameter. Based on these morphological characteristics, the causal agent was identified as Hyaloperonospora camelinae (1,3,4,5). DNA was extracted from mycelium and conidia collected from infected plants in two fields in the Savinja Valley (1HpC and 2HpC). Nuclear internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) were amplified by PCR assay from two isolates using the universal primers ITS4 and ITS5, and sequenced. Both samples yielded a 781-bp sequence, which showed 100% identity to H. camelinae ITS sequence JX445136 in GenBank. The nucleotide sequence was assigned to GenBank Accession No. KJ768405. Pathogenicity was confirmed by spraying 25 3-week-old plants of C. sativa cv. Ligena planted in pots (5 plants/pot) with a conidial suspension (105 conidia/ml) obtained from 10 infected plants of the same variety collected from the field 1HpC. Inoculated plants were covered with polyethylene bags for 2 days to maintain high humidity, and incubated at 20°C with a 12-h photoperiod/day in a growth chamber. Downy mildew symptoms first developed on leaves 6 days after inoculation. An additional 25 control plants sprayed with sterilized distilled water and otherwise treated similarly to the inoculated plants developed no symptoms. The identity of the pathogen on the inoculated plants as H. camelinae was confirmed based on the morphological features described above. Downy mildew of false flax caused by H. camelinae has been reported in Europe from Austria, Bulgaria, Germany, Poland, Portugal, Spain, and Switzerland (2); and in the United States from Florida, Oregon, Minnesota, Montana, Nebraska, and Washington (1,3,4,5). To the best of our knowledge, this is the first report of downy mildew caused by H. camelinae on C. sativa in Slovenia. The representative samples were deposited in the phytopatological herbarium of the Slovenian Institute of Hop Research and Brewing.

First Report of Thielaviopsis punctulata Causing Black Scorch Disease on Date Palm in Qatar.

Ceratocystis radicicola (anamorph: Thielaviopsis paradoxa) was reported as an economically important pathogen causing serious diseases on date palm such as rhizosis (2) and black scorch (3) or as an associated pathogen with diseased date palm (1). In this study, we report for the first time that C. radicicola also causes black scorch disease in Qatar. In April to May 2013, we conducted a disease survey in 11 farms located in northern and southern Qatar where three infected farms had an average of 10% disease incidence. Infected trees manifested different disease symptoms such as black scorch of leaves, inflorescence blight, and heart and bud rot. Infected tissues were surface sterilized with 1.0% NaOCl for 60 s, rinsed with distilled water, blotted dry, and then plated on potato dextrose agar (PDA) supplemented with 50 mg/liter Rose Bengal. Single fungal colonies were picked from hyphal tips and grown on PDA for 7 days at 25°C for further examination of the mycological characteristics. Colonies of five C. radicicola isolates on PDA developed aerial mycelium with a light gray color in culture plate, which later changed to black. Both light and scanning electron microscopy were employed to delineate species by spore morphology. Colonies produced ovate aleuroconidia (14 to 17 × 9 to 12 μm) and cylindrical phialoconidia (7 to 9 × 3 to 4 μm) characteristic features of C. radicicola. Phialoconidia (endoconidia) were hyaline to brown in chains produced from endoconidiophore, clamydospores (aleuroconidia), which were single with smooth or slightly rough wall. Additionally, C. radicicola produced single alueroconidia from conidiophores. Amplification of ITS rDNA region from fungal genomic DNA of five isolates, using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-CCTCCGCTTATTGATATGC-3') confirmed the isolated fungus as C. radicicola with no intra-specific variation among the fungal isolates. The length of ITS-rDNA sequence was 534 bp (KJ410228) and had 99 and 93% sequence identity with ITS-rDNA region from C. radicicola (HQ443203) and C. paradoxa (HC415073.1), respectively. A pathogenicity test was conducted using 3-year-old trees from three cultivars (Khalas, Khneezi, and Barhi) growing in sandy loam soil under greenhouse conditions (25 to 29°C and 12/12-h light/dark). Six trees from each cultivar were used for pathogenicity test, where three were inoculated and three other mock-inoculated. Eight millimeter diameter mycelial plugs were obtained from a C. radicicola culture on PDA medium and used to inoculate rachis region and basal petioles of date palm leaves with a 9-mm wound created with a cork borer. Control plants were mock-inoculated with PDA plugs. The inoculated area was covered with wet cotton to prevent dryness and the whole plant was covered for 72 h. Four days post infection (dpi), a rusty black infection appeared on the plants. Fifteen dpi, the whole leaf of inoculated stem showed typical symptoms, from which the fungus was re-isolated and colonies were maintained in PDA for morphological characterization, which were confirmed as C. radicicola. All trees from three cultivars showed symptoms with a variable severity from cultivar to another. To our knowledge, this is the first report of black scorch disease caused by T. punctulata in Qatar. This report highlighted the incidence of black scorch disease in Qatar demanding future research study to control the pathogen.

First Report of Powdery Mildew Caused by Erysiphe betae on Swiss Chard in China.

Swiss chard (Beta vulgaris L. subsp. cicla) is a widely planted vegetable in China. From May to June 2013, an outbreak of powdery mildew on Swiss chard cultivar Fangzheng was observed in the commercial fields in Zhoukou city of Henan Province, located in central China. More than 80% of the plants exhibited symptoms of the disease. At the beginning of infection, circular, white, dust-like colonies of powdery mildew occurred mainly on adaxial surfaces of leaves. As the disease progressed, white mycelia covered the foliar parts of plant. No cleistothecia were found on or in collected samples. Upon microscopic evaluation, conidiophores were unbranched with the length of 63 to 126 and width of 7 to 10 μm (n = 50), produced conidia singly, and composed of a cylindrical foot cell followed by one to three short cells. Conidia were colorless, hyaline, ovoid, measured 29 to 40 × 12 to 18 μm (n = 100), lacked fibrosin bodies, and produced germ tubes on the ends of the conidia. The fungus was identified as Erysiphe betae according to the morphological features (1). To verify the identity, the internal transcribed spacer (ITS) region was amplified with the universal primers ITS1 and ITS4 (2) and sequenced. The ITS sequence obtained was assigned as Accession No. KF268348 in GenBank, which showed 100% homogeneity with two ITS sequences of E. betae isolates from UK (DQ164432 and DQ164436). Koch's postulates were conducted by inoculating 15 healthy 5-week-old plants (cv. Fangzheng) with detached infected leaves, which grew in a growth chamber under 22/16°C (day/night), 50% relative humidity, 120 μmol/m2/s light and a 16-h photoperiod. Fifteen non-inoculated plants grew in another growth chamber with the same conditions as control. Symptoms consistent with the infected field plants were observed on the inoculated plants, while no symptoms were found on the control plants. Microscopic observation revealed that the pathogen growing on the inoculated plants was consistent with the morphology of the original fungus. To our knowledge, this is the first report of E. betae infection on Swiss chard in China (3).

First Report of Fusarium oxysporum and F. solani Causing Fusarium Dry Rot of Carrot in China.

Carrot (Daucus carota L.) is an economically important vegetable crop in China. In August 2008, a disease was observed on carrot in Inner Mongolia. The symptoms appeared as dry rot lesions on root surface, expressing light brown cankers with defined rounded or irregular shapes (1,3). The average disease incidence was up to 80% in Tuo Ke Tuo County. The disease has been a serious problem in these two counties since then, especially where consecutive carrot cropping was practiced. Carrot roots with typical dry rot symptoms were washed with tap water. Root tissues near the margin of necrotic lesions were excised, surface sterilized with 1% NaOCl for 3 min, and rinsed with sterile distilled water three times. The disinfected tissue was placed on potato dextrose agar (PDA) in a petri dish. Plates were incubated at 25 ± 1°C in the dark for 4 days. Fusarium single spore isolates were obtained from characteristic colonies (1). Three isolates (CF1, CF2, and CF3) were used for further study. The isolates were identified as Fusarium spp. on the basis of microscopic morphology on PDA. CF1 produced pink pigment, abundant falciform macroconidia of 14.7 to 38.2 × 4.5 to 5.7 μm with 2 to 3 septates, and elliptic microconidia of 7.5 to 15.1 × 3.3 to 5.4 μm with none or one septate. CF2 and CF3 produced light blue pigment, abundant falciform macroconidia of 16.4 to 34.4 × 4.0 to 6.1 μm with 2 to 3 septates, and elliptic microconidia of 6.7 to 10.7 × 3.0 to 4.9 μm with none or one septate. They were further identified and confirmed by PCR. The PCR involved amplifying the internal transcribed spacer (ITS) region of ribosomal DNA using genomic DNA as the template with universal primers ITS1 and ITS4 (2). The PCR products were sequenced. BLAST analysis of these sequences against the GenBank database determined the taxonomy of the isolates. The sequence of CF1 was 99% identical to F. oxysporum (Accession No. KC594035); sequences of CF2 and CF3 were 99% identical to F. solani (KC215123). To confirm the pathogenicity of the isolates, mature carrot roots (cv. Hong Ying 2) were inoculated with mycelial plugs (5 mm in diameter) cut from the margin of actively growing colonies on PDA plates. One mycelial plug was placed on each carrot root, with the mycelial side facing the root. PDA plugs were used for controls. Each treatment had five replicates. The inoculated roots were incubated in a humid chamber (90% RH) at 25°C. Four days after incubation, mycelia of the isolates developed and covered most of the surface of carrot roots, and brown rot lesions were observed on all inoculated roots, while the controls remained symptomless. This experiment was repeated. In another trial, carrot seeds (cv. Hong Ying 2) were sown in sterilized soil in pots (30 × 25 cm opening) with 15 seeds per pot. The soil was infested with either CF1, CF2, or CF3 by adding spore suspension to make the final concentration of 1 × 104 CFU/g soil. Plants grown in non-infested soil served as controls. There were three replicates per treatment. All the treated pots were placed in a field. After 13 weeks, the same symptoms of dry rot were observed as previously described. No symptoms were observed on the control plants. The trial was repeated. Symptomatic tissues from the inoculated roots were sampled and the pathogen was re-isolated, and identified using PCR. To our knowledge, this is the first report of F. oxysporum and F. solani causing dry rot of carrot in China.

First Report of Apple Bitter Rot Caused by Colletotrichum godetiae in the United Kingdom.

Apple is an important crop in United Kingdom, with a total production of 233,750 tonnes in 2011. Symptoms of apple bitter rot were observed on apple fruits (Malus domestica L.) in the Newcastle area, United Kingdom, in October 2008. Lesions were round, 1 to 5 cm in diameter, brown and dry, with acervuli producing yellowish spore masses in concentric bands. Infected material was sent to the W-HRI (University of Warwick) for identification of the causal agent. Fungal isolates with morphological characteristics similar to those of Colletotrichum acutatum sensu lato were isolated from diseased fruits. Monoconidial isolates were grown on PDA at 25°C with a 12-h light period. The cultures were light gray, with cottony aerial mycelium getting darker with age and with color ranging from whitish to dark gray on the reverse side of the colony. The cultures have yellowish spores masses and dark melanized structures similar to acervuli. Colletotrichum spp. are difficult to identify solely on morphology; therefore, representative isolates were used for multi-locus gene sequencing and characterization (1). Genomic DNA was extracted using a modified Chelex100 protocol. Three loci were amplified and sequenced: the ITS region was amplified and sequenced using the universal primers ITS4 and ITS5. Primers TB5 and TB6 were used for the amplification and sequencing of the variable region of the TUB gene. Primers GDF1 and GDR1 were used to amplify a 200-bp intron region of the GAPDH gene. No differences were found among the strains at any of the loci. One sequence for each locus has been deposited in GenBank under accessions KF834206 (ITS), KF834207 (TUB), and KF834208 (GAPDH). In GenBank, ITS sequences matched with 100% identity to C. higginsianum (EU400147) and to C. gloeosporioides (AJ301931 to 972); and with identity between 99.6 and 99.8% with sequences belonging to C. godetiae (part of C. acutatum species complex). The TUB sequences match with 100% identity to more than 25 sequences belonging to C. godetiae. The GAPDH sequences match with 100% identity to JQ948739 and 35 belonging to C. godetiae strains IMI 381927 and CBS 131331. A multilocus phylogenetic tree (ITS, TUB, and GAPDH) was reconstructed using sequences of reference strains belonging to C. higginsianum, C. gloeosporioides, C. godetiae, and related species. The phylogenetic tree confirmed the identity of the strains isolated from apple as C. godetiae. Koch's postulates were tested with representative isolate by artificial inoculation of 12 healthy fruits of the cv. Golden Delicious. Fruit surfaces were sterilized with 70% ethanol, wounded with a sterile needle, and then inoculated with a plug of actively growing mycelium prepared from a 10-day-old culture grown on PDA. Inoculated fruits were incubated in sterile conditions at 25°C with a 12-h photoperiod. In 83% of fruits, symptoms appeared between 7 and 15 days later. The rot begins as light brown, circular lesion getting darker with orange spore masses. Fungal colonies isolated from the lesions and cultured on PDA have identical morphological characteristics of the isolate used for the pathogenicity assay. To the best of our knowledge, this is the first report of apple bitter rot caused by C. godetiae in the United Kingdom. Apple bitter rot is spread worldwide and in moist, temperate regions it is considered one of the most important diseases causing considerable crop losses. Since the losses are more severe under prolonged warm and wet weather conditions, bitter rot caused by C. acutatum species may become an emerging problem in the United Kingdom in the near future, and may require investigation of management practices to control this new disease.

First report of Colletotrichum circinans causing smudge on onion in Iran

In October and November 2012, white-scaled onions bulbs with necrotic symptoms were collected in the area under cultivation in Khuzestan province. Symptoms were dark spots frequently organised in concentric rings, forming patches of up to 1 cm in diameter (Fig. 1). These spots are made up of stromatic masses formed beneath the cuticle of the host, consisting of intertwining thick-walled mycelial threads in dark green to black stromata, usually only a fraction of a millimetre in diameter and a few to several hundred microns thick. The affected tissue was surface sterilised with sodium hypochlorite (0.8% chlorine) for 30 seconds and dried with sterile paper before small pieces were excised and aseptically transferred to potato dextrose agar (PDA) and water agar (WA) in petri dishes and incubated in the dark at ambient temperature (30 ± 2°C). Isolates grown on PDA, produced acervuli with dark, almost black, 1-3 septate acicular setae having a bulbous base, measuring up to 290 times 8 üm (Fig. 2 A, B). Conidia were hyaline, unicellular, 16-25 times 3-5 üm, slightly falcate, guttulated (Fig. 1 C). A small amount of mycelia was scraped from a seven-day-old culture and the internal spacer region (ITS) of the extracted fungal DNA was amplified with universal primers ITS1 and ITS4. The resulting sequence (509 bp) was submitted to the NCBI GenBank (isolate IRAN2185C, Accession No. KJ458968,). A blast search of ITS sequences revealed that this fungus was Colletotrichum circinans with 100% query coverage with KC790955 and AJ301955 and 99% query coverage with GQ369595. Diseased tissue was cut into 1-2 cm lengths, surface-sterilised, and the original pathogen from onion placed on water agar to recover the fungus. Three isolates of C. circinans were tested for pathogenicity to onion bulbs by artificial inoculation with conidial suspensions (1-2 times 10 conidia/ml) that were prepared from 20-day-old PDA cultures. Sterile distilled water was used as control. Plastic boxes containing the inoculated material were placed in an incubator at 26°C for five days. Disease ratings were made based on the degree of anthracnose symptoms induced five days after inoculation. The inoculation test was performed in three independent replicates. C. circinans was re-isolated from these lesions fulfilling Koch's postulates. C. circinans was previously recorded as C. dematium (Pers.) Grove f. circinans Arx, which is specific to Allium spp. (von Arx, 5; von Arx, 6). However, Sutton (4) described C. circinans as a distinct species from C. dematium. The fungus causes smudge or leaf spot of Allium spp. (Farr et al., 2) and has been reported from Korea, Japan, China, Argentina, India, UK and most other regions of the world (Cho & Shin, 1; Kiehr et al., 3). This the first report of C. circinans in Iran. A culture of C. circinans was deposited in the Iranian Research Institute of Plant Protection (IRAN2185C).

First Report of Root Rot of Watermelon Caused by Ceratobasidium sp. in Sonora, Mexico.

Watermelon is one of the major crops grown in Mexico and represents 4% of the total cultivated area with fruits in this country. In 2013, Sonora State was ranked second in the production of watermelon at a national level. Fungal and oomycete diseases are among the main biotic factors affecting watermelon production, particularly those caused by species of the genera Fusarium, Phytophthora, Pythium, and Rhizoctonia. During the spring of 2013, wilting or death symptoms were confirmed in approximately 50% of ungrafted watermelon plants grown in four sampled fields along the coast of Hermosillo and Guaymas Valley in Sonora, Mexico. On both roots and stems of infected plants, localized lesions were found that were 0.2 to 2.0 cm long, reddish brown, and slightly sunken on the stem base. In some cases, the discolorations encompassed nearly 90% of the root system. One-centimeter pieces from the edge of lesions on stems and roots were superficially disinfected with 1% sodium hypochlorite, then rinsed with sterile distilled water, placed onto petri dishes containing potato dextrose agar (PDA), and incubated at 25°C for 3 days. Fungal colonies were white initially, then turned brown, and septate hyphae were 3.7 to 4.3 μm in diameter and branched at right angles with a constriction at the origin of the branch point. These characteristics are typical of the genus Rhizoctonia. Binucleate cells from five isolates were observed using a lactophenol aniline blue solution stain, according to Ceratobasidium morphological descriptions. Mycelia from five isolates grown on PDA was used for DNA extraction. The rDNA-ITS region was amplified using PCR with the universal fungal primers ITS1 and ITS4 (3). The purified products were separately sequenced in both directions using the same primer pair. The sequences obtained were 99% similar to those of Ceratobasidium sp. AG-F and AG-Fa isolates (accessions KC193238.1 on Tagetes erecta, HQ168370.1 on Musa spp., and JX913821.1 on soy-rice-weeds, respectively) from GenBank (2,4). The pathogenicity of the fungus was tested under growth chamber conditions. Sets of seven healthy watermelon seedlings of the Sugar red variety were inoculated with five isolates of Ceratobasidium. Three disks (8 mm in diameter) of mycelia grown on PDA were placed around the roots of each plantlet. The pots were maintained at 27 ± 0.1°C for 14 days with a photoperiod of 12 h. Seven uninoculated seedlings were used as a control. Initial symptoms showing water-soaked lesions developed on all inoculated seedlings within 6 to 7 days, while typical disease symptoms appeared after 10 to 14 days after inoculation. Seedlings without inoculum were free from infection. The fungus was re-isolated from the inoculated seedlings on PDA, and identified as Ceratobasidium sp., confirmed using morphological characteristics. A similar disease has been reported recently in Italy and Arizona (1); however, this report is the first description of a Ceratobasidium sp. causing root rot of watermelon in Sonora, Mexico. Agricultural areas where the study was carried represent 90% of the total area cultivated with watermelon in this state, so it is necessary to evaluate the impact of this pathogen in the crop.