Mycelial Research Articles

FIRST REPORT OF ASSOCIATION OF SCLEROTIUM CEPIVORUM WITH WHITE ROT OF GARLIC IN OMAN

In the spring of 2014, a disease of garlic (Allium sativum) was observed in more than 30 plots in Jabal Akhdar (Sultanate of Oman), which was characterized by premature yellowing and wilting of the leaves followed by plant death. Upon uprooting, white cottony mycelium and black sclerotia were observed on the outer layer of the bulbs, roots and surrounding soil. The infection appeared to progress from the outer layers of the bulbs to the inner ones so as to involve the whole organ. Disease incidence ranged from 20 to 100%. On potato dextrose agar (PDA), fungal isolates with whitish/grayish mycelium and small-sized sclerotia typical of Sclerotium cepivorum were consistently recovered from symptomatic bulbs and roots. The sequence of the internal transcribed spacer (ITS) region of the ribosomal DNA (Al-Sa'di et al., 2007) of three fungal isolates using ITS1 and ITS4 primers produced in all cases a nucleotide sequence 510 bp long (accession Nos GCC3471-73) which, when compared with sequences from database revealed 99.8% nucleotide similarity to a previously published sequence (accession No. FJ460433) of the garlic isolate H-292 of S. cepivorum from Hungary. The disease was reproduced in healthy garlic bulbs planted in inoculated soil with an incidence ranging from 30 to more than 65%. To the best of our knowledge, this is the first report of the association of S. cepivorum with white rot of garlic in Oman.

بررسی تاثیر اسانسهای ریحان و مریم گلی کبیر بر رشد لیستریا مونوسیتوژنز و آسپرژیلوس فلاووس در پنیر سفید ایرانی

با توجه به تاثیرات منفی نگهدارنده های شیمیایی بر سلامت مصرف‌کنندگان، توجه مراجع قانونی و صنایع غذایی بر کاربرد اسانس‌ها و عصاره‌های گیاهی بعنوان نگهدارنده‌های طبیعی در مواد غذایی متمرکز شده است. در این پژوهش تاثیر اسانس‌های ریحان و مریم گلی کبیر بر رشد لیستریا مونوسیتوژنز و آسپرژیلوس فلاووس طی دوره نگهداری پنیر سفید ایرانی مورد مطالعه قرار گرفت. روش‌های بکار رفته در این تحقیق مشتمل بر تعیین حداقل غلظت مهارکنندگی رشد (MIC)، حداقل غلظت باکتری‌کشی (MBC) و حداقل غلظت قارچ‌کشی (MFC) اسانس‌ها، تعیین تاثیر غلظت‌های مختلف اسانس‌ها بر رشد لیستریا و آسپرژیلوس در پنیر طی دوره نگهداری محصول بود. ترکیبات اصلی اسانس مریم گلی کبیر شامل لینالیل استات، لینالول و اسانس ریحان شامل لینالول و آلفا-کادینول بود.MIC و MBC اسانس مریم گلی، به ترتیب، معادل 015/0% و 02/0% و اسانس ریحان برابر با 05/0% و 06/0% برای لیستریا بود. همچنین، MIC و MFC در برابر آسپرژیلوس برای اسانس مریم گلی معادل 5/0% و 65/0% و برای اسانس ریحان برابر با 6/0% و 8/0% بدست آمد. غلظت 35/0% اسانس مریم گلی و 5/0% اسانس ریحان از تولید اسپور توسط قارچ در محیط کشت جلوگیری نمود. اسانس مریم گلی در غلظت 1% طی دوره نگهداری پنیر از رشد آسپرژیلوس بطور کامل بازداری نمود و جمعیت لیستریا را نسبت به شاهد log 6 کاهش داد. اسانس ریحان تاثیر ضدمیکربی ضعیف‌تری نسبت به اسانس مریم گلی کبیر نشان داد.

Enhancement of Neuromuscular Activity by Natural Specimens and Cultured Mycelia of Cordyceps sinensis in Mice.

The present study was aimed to evaluate the effect of natural specimen and laboratory cultured mycelia of Cordyceps sinensis on neuromuscular activity in mice. The powder of natural specimen and laboratory cultured Cordyceps sinensis was orally administered at the dose rate of 100, 300 and 500 mg/kg for 30 days. Natural specimen and in vitro propagated Cordyceps sinensis showed significant (P<0.05) enhancement in neuromuscular endurance and antidepressant activity at 300 and 500 mg/kg as compared to the control group. However, the fungus did not proved to be as effective as fluoxetine in exhibiting antidepressant action. Muscular endurance was determined on a Rota rod apparatus while antidepressant (mood elevating) activity was measured on a photoactometer in Swiss albino mice. The effects produced by both natural specimens and laboratory cultured Cordyceps sinensis were comparable and showed almost equal potency.

Alternative control of early blight of tomato using plant extracts from Acacia nilotica, Achillea fragrantissima and Calotropis procera

The in vitro and in vivo antifungal potential of extracts of three wild medicinal plants, ( Acacia nilotica (L.) Delile , Achillea fragrantissima (Forssk.) Sch.Bip. and Calotropis procera (Aiton) W. T. Aiton) was examined against Alternaria solani , the causal agent of the early blight of tomato . Aqueous or ethanol extracts of all tested plants reduced the mycelial growth and conidium germination of A. solani in vitro . Ethanol extracts were more effective against the pathogen than the aqueous extracts. Extract of C. procera exhibited more antifungal potential against the pathogen than other plant extracts. Observations by scanning and transmission electron microscopy showed dramatic alterations in the morphology and ultrastructure of A. solani when treated with the ethanol extract of C. procera at a concentration of 20%. Phytochemical screening confirmed the presence of many bioactive constituents in the extracts which were in greater amounts in C. procera than the other two plants. In a plot experiment, both types of extracts from C. procera reduced disease severity. Tomato fruit yield was increased after the treatment with the plant extracts.

FIRST REPORT OF PLEOSPORA TARDA CAUSING LEAF SPOT OF LEPIDIUM DRABA IN IRAN

Since 2009, leaf spot symptoms were observed on Lepidium draba (whitetop) an invasive weed collected in wheat plots (Triticum sp.) in the Hamadan Province of Iran. Typical symptoms included chlorosis of the basal leaves with black discrete ascomata protruding from the leaf surface. Colonies of a fungus recovered from diseases leaves were brown to olivaceous with a white aerial mycelium in the center. Since the morphological characters of this mycete were consistent with the description of Pleospora tarda (Simmons, 1985), its identification was confirmed by amplifying and sequencing the internal transcribed spacer region (ITS) of a representative isolate (GenBank accession No. KC660992). To fulfill Koch’s postulates, a spore suspension (105 ascospores/ml) of the fungus was sprayed onto leaves of whitetop seedlings at the 4-6 leaf stage. Inoculated seedlings were maintained in a growth chamber at 25°C, with more than 75% relative humidity and a 12 h light photoperiod. Inoculated plants showed symptoms like those observed in the field 14 days post inoculation. P. tarda was reisolated from inoculated plants and a culture was deposited in the Iranian Research Institute of Plant Protection with the accession code IRAN 2184C. To our knowledge, this is the first report of P. tarda infecting whitetop in Iran and in the world.

FIRST REPORT OF A LEAF SPOT DISEASE CAUSED BY SCLEROTIUM ROLFSII ON JASMINIUM MULTIFLORUM IN INDIA

Jasminium multiflorum is an important crop grown ex- tensively in parts of Southern India. A characteristic leaf spot disease was observed during field surveys conducted in 2013-2015. The disease incidence ranged from 18 to 23% over about 45 cropped hectares. Water-soaked lesions (2-8 mm) appeared initially on the basal leaves followed by the development of large necrotic spots (0.5-1.5 cm) with sclero- tial bodies at the center of the necrotized areas. Affected leaf tissues were surface-sterilized with 2% NaOCl, transferred onto potato dextrose agar (PDA) and incubated at 28±2°C. Fungal colonies with dense, aerial whitish cottony mycelium with uniformly globoid sclerotia (1-2.2 mm) were observed after 10-12 days of incubation. Based on the morpho-cultural characteristics, the fungus was identified as Sclerotium rolfsii (Mordue, 1974). The identification was confirmed by PCR amplification of ITS-rDNA using ITS1/ITS4 primers (White et al., 1990). The PCR product was sequenced directly and the sequence analysis revealed 100% homology with S. rolf- sii (GenBank accession No. KP412469.1). A representative sequence of S. rolfsii was deposited in GenBank (accession No. KT768140.1). Pathogenicity tests were conducted on 30 healthy leaves by inoculating 2-3 sclerotia from 12 days-old culture. The appearance of necrotic leaf spots was noticed on 22 inoculated leaves seven days post inoculation. No such symptoms were observed on control leaves challenged with water. The fungal pathogen was re-isolated on PDA and its identity confirmed. To the best of our knowledge, this is the first report on the occurrence of S. rolfsii causing leaf spot of J. multiflorum in India.

FIRST REPORT OF FRECKLE DISEASE OF BANANA CAUSED BY PHYLLOSTICTA CAPITALENSIS IN GUANGXI, SOUTHWEST CHINA

In December 2013, irregular spots with gray centers, dark brown edges and chlorotic halos were observed on the old leaves of bananas of cv. Williams (AAA, Cavendish) in Guangxi province (China), with an incidence of 15% to 30%. Small pieces of diseased leaf tissues were surface-sterilized in 75% ethanol for 30 s followed by 2 min in 0.1% HgCl2, rinsed in sterile water, plated on potato dextrose agar (PDA) and incubated at 28oC in darkness. A single-spore culture had initially an olive or dark green mycelium with granular surface and white edges which, over time, turned almost black. Asci containing 8 ascospores, were clavate or cylindrical, stipitate, and measured 35.6-43.6×8.5-10.6 μm. Conidia measured 7.2-11.8×4.0-6.3 μm, and were obpyriform or nearly elliptic. Single spore cultures of the isolate were obtained and used for molecular identification. The internal transcribed spacer (ITS) region of rDNA was amplified and sequenced by utilizing the primers ITS4/ITS5 (2). The ITS region of the isolate (Accession No. LM994823) was 629 bp in length. BLAST analysis of the sequences showed a 99% homology with two Phyllosticta capitalensis isolates (accession Nos KM979840 and JQ743587). Pathogenicity was conducted using healthy banana plants with five or six leaves, approximately 30 cm in height. Nine plants were used for each treatment and three leaves of each plant were artificially inoculated. Mycelial plugs were placed on the surface of the leaves, whereas controls were treated only with agar plugs. All the treated plants were covered with plastic bags for 2 days and incubated at 28oC, 75% relative humidity (RH) and with a 12-h photoperiod. After 7 days, typical lesions identical to those observed on the field-grown plants appeared on the inoculated plants, whereas control plants remained healthy. The re-isolated fungus was identified as P. capitalensis by morphology and molecular analysis. P. capitalensis is generally considered an endophyte or saprophyte, within a wide host range and distributed in different plants (Wulandari et al., 2010; Wong et al., 2012). However, it is a weak plant pathogen which was the causal agent of plant diseases in few cases (Wikee et al., 2013). To our knowledge, this is the first report of P. capitalensis causing freckle disease of banana in China.

BOTRYTIS CINEREA, NEW PATHOGEN INFECTING OREGANO CROPS IN ARGENTINA

In October 2013 plants with necrotic leaves and cankers on the stems were observed in oregano crops (Origanum vulgare ssp. hirtum) at Tres Esquinas (Mendoza, Argentina). The necrosis had a basipetal progress, affected the center of the bush and, in some cases, resulted in the death of the plant. Following isolation on potato dextrose agar (PDA), effuse greyish to brown colonies developed, which produced irregular black sclerotia. The mycelium was branched, septate, hyaline to brown-coloured and produced conidiophores bearing one-celled, egg-shaped and hyaline conidia, grouped in glistening heads. Based on these morphological traits, the fungus was tentatively catalogued as Botrytis sp. (Ellis, 1971), until molecular identification was performed. Its pathogenicity was tested by inoculating oregano stems with agar plugs from fungal colonies, while control plants were inoculated with sterile agar plugs. The plants were covered with a plastic bag for 24 h. After 5 days, the field syndrome was reproduced in inoculated plants, which showed an incipient necrosis of the stem and wilting of the leaves. From these plants the pathogen was successfully re-isolated, fulfilling Koch’s postulates. Molecular identification was carried out by PCR amplification using the primer pair ITS1/ITS4 (White et al., 1990). The amplified product was purified, sequenced (GenBank accession No. KT921335) and compared with the equivalent sequences from database. A complete match score was found with Botrytis cinerea, confirming the identity of the pathogen. B. cinerea was recorded from Italy as a pathogen of Origanum majorana (Pensa et al., 2007). To the best of our knowledge this is the first report of B. cinerea affecting oregano crops in Argentina.

FIRST REPORT OF NECTRIA HAEMATOCOCCA ASSOCIATED WITH DIEBACK OF OLIVE TREES IN TUNISIA

During a routine survey for olive diseases conducted in autumn 2013 in southern Tunisia (Bir Ali region), wilting and chlorosis of the leaves accompanied by brown-to-black discoloration of the wood in cross-sectioned twigs were observed on 3- to 10-year-old olive trees. On potato dextrose agar (PDA), a fungus was isolated from symptomatic twigs and branches with an initially white mycelium that over time became light gray-brown. This fungus was identified as Nectria sp. based on morphological characteristics and analysis of the ITS gene region (White et al., 1990). A BLAST search of GenBank database revealed 99% homology of the amplified product with a reference sequence of Nectria haematococca (strain HLJ_14, accession No. JN088237.1). Pathogenicity tests were conducted on 10 two-year old olive trees of cv. Chemlali, by placing a mycelial plug in a shallow wound on the stem of each plant. Control plants were inoculated with sterile PDA plugs. Two months post inoculation, symptoms appeared, with stems showing brown discolorations and necrotic lesions. Controls remained healthy. N. haematococca was recovered from necrotic lesions, thus fulfilling Koch's postulates. N. haematococca was held responsible for root rot of olive trees in Argentina (Barreto et al., 2003). To our knowledge this is the first report of N. haematococca as a causal agent of dieback of olive trees in Tunisia.

GENETIC DIVERSITY OF SCLEROTINIA SCLEROTIORUM WITHIN A SINGLE SUNFLOWER FIELD IN WENQUAN, XINJIANG PROVINCE, CHINA

To unravel the genetic diversity of Sclerotinia sclerotiorum, 90 isolates collected from a single sunflower field in Wenquan of Xinjiang were studied using the method of mycelial compatibility grouping (MCG). Meanwhile, the oxalic acid (OA) secretion ability, polygalacturonases (PG) enzymatic activity and pathogenicity of the isolates within the same and between different MCGs were studied. The distribution of inversion minus (Inv-) and inversion plus (Inv+) isolates of S. sclerotiorum at MAT locus within the same MCGs and also in the whole population were identified using specific PCR primers. The sensitivity of S. sclerotiorum isolates to both carbendazim and dimethachlon fungicides were also tested in this study. The results suggested that the 90 isolates from a single sunflower field in Wenquan, Xinjiang region, can be grouped into 15 MCGs. MCG1, the biggest group, contained 34 isolates, representing 37.8% of all the tested isolates; 6 MCGs contained only a single isolate; 8 MCGs include 2 to 13 isolates each. The dramatically variation on OA level, PG enzyme activities and pathogenicity was observed among isolates from the same and between different MCGs, and no obvious positive correlation was detected between OA, PG and pathogenicity. For mating type distribution ratio, 17:17 (1:1) ratio of Inv+ and Inv- isolates at mating type locus (MAT) was observed only in MCG1, however, 12:1, 7:2 and 9:1 of Inv+ to Inv- ratio at MAT locus was detected in MCG2, MCG4 and MCG6, respectively. The distribution ratio of Inv+ and Inv- in the whole population is 56:34, which is deviated from the 1:1 steady state. The results of fungicide sensitivity test within MCG1 and MCG2 suggested that 84% of isolates is more sensitive to dimethachlon than to carbendazim. Seven isolates, three from MCG1 and four from MCG2, showed the opposite tendency of sensitivity to both fungicides. Three isolates, one from MCG1 and two from MCG2, showed the same sensitivity to both fungicides. Based on above results, we concluded that dramatic genetic diversity do exist within S. sclerotiorum population collected from a single sunflower field in Wenquan of Xinjiang.

FIRST REPORT OF PHYTOPHTHORA NICOTIANAE ON CATHARANTHUS ROSEUS IN BANGLADESH

Annual vinca (Catharanthus roseus), also known as Madagascar periwinkle, is native to the island of Madagascar, and now common in many tropical and subtropical regions worldwide, including Bangladesh (Chevallier, 1996). It has long been cultivated for herbal medicine and as an ornamental plant. Symptoms of stem canker and leaf blight developed on C. roseus in a landscape planting at Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh, in June, 2014. To identify the pathogen, infected leaves were placed in water for 48 h, then the mycelia were transferred onto corn meal agar plates. Mycelial growth was patchy and irregular, coenocytic and tufted with an arachnoid branching habit, consistent with descriptions of Phytophthora nicotianae. The pathogen was also cultured on PARP medium (Ferguson and Jeffers, 1999) and V8 agar medium and pure cultures were established. Mycelial blocks (6 mm in diameter) were placed in sterile water for 3 days, then at 5 C for 15 min in the dark to stimulate production of sporangia and zoospores. Molecular identification was done by amplifying the ITS region of nuclear rDNA using ITS 4 and ITS 6 primer pairs. BLAST search revealed that the isolate had 100% identity with P. nicotianae (GenBank accession No. KT175508.1). The nucleotide sequence was deposited in GenBank under accession No. KU244700. Pathogenicity tests were conducted by inoculating Catharanthus leaves, previously wounded by pricking with a sterile needle, with 50 μl and 100 μl aliquots of zoospores. Foliar symptoms consistent with blight observed in the field developed on all the inoculated leaves after 72 h. Koch’s postulates were fulfilled by re-isolating the pathogen from the lesions on the inoculated leaves. To our knowledge, this is the first molecular identification of P. nicotianae on Catharanthus in Bangladesh.

Identifying resistance to Verticillium wilt in local Spanish olive cultivars

The resistance of 42 Spanish olive cultivars to Verticillium dahliae was evaluated in two experiments carried out in two consecutive years, conducted under greenhouse conditions. In both experiments, bare root systems of 5-month-old plants were inoculated with a semisolid mass of a mixture of culture medium and conidia and mycelium of the fungus. The cultivars Frantoio and Picual were used, respectively, as resistant and susceptible reference cultivars. All cultivars were evaluated on the basis of final values of the area under the disease progress curve, mean severity of symptoms and percentage of dead plants. Most of the tested cultivars were susceptible to Verticilium wilt. However, eight genotypes (‘Cornezuelo de Jaen’, ‘Verdial de Badajoz’, ‘Jaropo’, ‘Negrillo de Estepa’, ‘Jabaluna’, ‘Ocal de Alburquerque’, ‘Asnal’ and ‘Racimal’) exhibited resistance to the disease.

Effection of Anti-fatigue of Selenium-rich Cordyceps Militaris in Mice

Purpose to probe into the anti-fatigue ability of selenium-rich Cordyceps militaris. Research methods: (1) Determination of mycelium components: The polysaccharides, organic selenium and superoxide dismutase of mycelium were measured; (2) Determination of anti-fatigue ability: The mice were treated with different dosages of selenium-rich mycelium per day. Each group was tested separately of the mice’s exhaustive swimming time and the content of blood lactate and blood urea nitrogen. Results: the content of mycelia polysaccharide and the activity of superoxide dismutase (SOD) in Cordyceps militaris enriched selenium mycelia was increased. The mice treated with mid-dosage obviously prolonged the weight loading swimming exhaustive time. The mid-dosage of selenium-rich Cordyceps militaris could prolong or reduce the production of lactate acid, quicken the clearance of lactate acid. Conclusions: The polysaccharides content, organic selenium content and the activity of SOD enzyme in the selenium-rich mycelium powder increased significantly. selenium-rich Cordyceps militaris can increase the anti-fatigue ability of the exercise in the mice.

First report of Curvularia spicifera causing brown rot of citrus in southern Italy.

In 2014, rot symptoms were observed on mandarin fruits (Citrus reticulata) in a grove of Metaponto (southern Italy). Five to 7 mm lesions, colonized by a brown mycelium characterized diseased tissues. Fruit was collected, and surface-sterilized in 2% sodium hypochlorite. Tissue portions, removed in between symptomatic and healthy areas, were placed on potato dextrose agar (PDA) selective for yeasts and fungi and incubated at 24°C. The obtained fungal isolate was deposited in the collection of DISSPA, University of Bari, Italy, in its monoconidial form. On PDA, colonies were fast growing, initially light brown, turning dark brown after 7 days, raised and lobate-undulate. The aerial mycelium was cottony. Conidia were 18.5±3 μm long × 7±3.4 μm wide, ellipsoidal or oblong, straight, with rounded ends, light brown, 3-transverse septated, with an evident hilum and walls fairly thick, thinner towards the ends. Conidiophore light to medium brown, of variable length, scattered or clustered, often branching were observed. Based on these morphological characters, the fungus was identified as Curvularia spicifera (Bainier) Boedijn. Curvularia is a hyphomycete associated with over 60 host genera (Manamgoda et al., 2011). Most Curvularia spp. are found in tropical regions, though a few have been reported in temperate zones. A molecular identification was also performed. Internal transcribed spacer regions ITS1 and ITS2, including the 5.8S gene, were amplified using the universal primers ITS5/ITS4, as reported by White et al. (1990), and sequenced (GenBank Accession N°KR229979). Blast analysis revealed 100% nucleotide similarity with other ITS sequences of C. spicifera available in Genbank. To fulfill Koch’s postulate, a pathogenicity test was conducted on mandarins. Fruit was surface sterilized with 2% sodium hypochlorite solution, rinsed with sterile water, dried, and wounded once at the equator with a sterile nail, by making a 3×2 mm injury that was then inoculated by the spore suspension (105 conidia/ml). Fruit was placed at 24°C and 95% RH. Non-inoculated fruit was used as a control. Seven days after inoculation, fruit showed symptoms similar to those previously observed in the sampled fruit, while control fruit showed no symptoms. The colonies isolated from infected tissues showed the same morphological and molecular features of the identified strain. To our knowledge, this is the first report of citrus fruit infection by C. spicifera in Southern Italy. Further studies will be conducted to determine the incidence of the disease, as well as possible control means, since rot symptoms would make the fruit unmarketable.

FIRST REPORT OF LAUREL ANTHRACNOSE CAUSES BY COLLETOTRICHUM ACUTATUM IN CENTRAL ITALY

Laurus nobilis (laurel) is an evergreen tree whose communities are spread throughout Italy (Filibeck, 2006). During summer 2014, in a laurel hedge of a garden near Rome symptoms were seen consisting of shoots with tips with typical 'shepherd’s crook' and necrotic spots on leaves and twigs. Tissue fragments from the margin of necrotic spots were cut, surface-disinfected (1% NaClO for 1 min), plated onto potato dextrose agar (PDA) and incubated at 25°C in the dark. A single fungal species was consistently isolated, pure cultures of which were obtained by single conidia transfer. The fungus was morphologically identified as Colletotrichum acutatum. DNA of one monosporic isolate (2002ER) was extracted and amplified with primers specific for the internal transcribed spacer (ITS) regions and the beta-tubulin gene (TUB2). This isolate showed 100% similarity with several C. acutatum strains (i.e. AJ301964, AJ301950, DQ286130) for the ITS region and C. acutatum strains (i.e. JQ424939, JN121284) for the TUB2 gene. Our sequences were deposited in European Nucleotide Archive (ENA) with the accession No. LN836025 for ITS, and No. LN836026 for TUB2. Mycelial plugs (5 mm diameter) cut from the margins of actively growing cultures were inserted under the epidermis at different levels of six-year-old potted laurel plants. Controls were inoculated with sterile PDA plugs. After 45 days inoculated plants grown at 24±2°C showed symptoms similar to those observed in the garden and C. acutatum was re-isolated from the margin of the lesions. There are a few report of anthracnose by other Colletotrichum species on laurel in Europe (Constantinescu and Jonsson, 1987; Gore and Bucak, 2006). To our knowledge, this is the first report of damages caused to laurel in Italy by C. acutatum.

FIRST REPORT OF LEAF SPOT CAUSED BY COLLETOTRICHUM COCCODES ON SALVIA GREGGII IN ITALY

During summer 2014, leaf spots were observed on a number 6- to 8- month-old plants of autumn sage (Salvia greggii) in a private garden near Biella (northern Italy). Brown small irregular lesions were present on the leaves of plants grown in the shadow with high relative humidity which, at the final stage, were almost completely defoliated. A fungus consistently isolated on potato dextrose agar (PDA), was characterized by a gray mycelium producing hyaline, cylindrical, aseptate and thin walled conidia (7.5-19.3 x 2.8-6.0, average 13.9 x 4.4 μm) in acervuli which, based on these traits, was identified as a Colletotrichum sp. (Bailey and Jeger, 1992). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4, and custom-sequenced. BLAST analysis (Altschul et al., 1997) of the 510 bp amplicon (GenBank accession No. KP792748) showed 100% similarity with Colletotrichum coccodes (AM422215.1). Leaves of three healthy 10-month- old potted plants of S. greggii were inoculated by spraying a conidial suspension of 1x105 CFU/ml. Control plants were sprayed with sterile distilled water. Plants were covered with plastic bags and kept in a greenhouse at 18 to 25°C. Symptoms similar to those observed on naturally infected sage appeared on all inoculated plants 14 days post inoculation while controls remained healthy. C. coccodes was consistently reisolated only from inoculated plants. This is to our knowledge the first report of C. coccodes on S. greggii worldwide. The disease is currently limited to the area where it was first observed. However, C. coccodes could become a significant problem due to its wide host range that includes also other member of lamiaceae such as peppermint.

First report of powdery mildew caused by Erysiphe alphitoides on Quercus rugosa in Mexico.

During September to November of 2013 and 2014, typical symptoms of powdery mildew were detected on the leaves of netleaf oak (Quercus rugosa) trees growing in the Distrito Federal (Mexico). Symptoms appeared as irregular necrotic lesions with greyish powdery masses on both sides of the leaves. The preliminary identification of fungus was done based on of morphological characters. Mycelium was amphigenous, persistent, greyish, in dense patches or effuse. Hyphal appressoria were lobed and solitary. Conidiophores were hyaline, erect, straight, length up to about 86 μm. Foot cells were cylindrical, straight, followed by 1-3 shorter cells, and forming conidia singly. Conidia were hyaline, obovoid-doliiform, 27-37x16-24 μm, with germ tubes terminal to subterminal. Conidial appressoria were lobed. Chasmothecia were not observed. The morphological characters were consistent with those of the anamorphic state of Erysiphe alphitoides (Braun and Cook, 2012). To confirm the identification, the complete internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4 and ITS5, and sequenced directly. The resulting 647 bp sequence was deposited in GenBank (accession No. KR269919). A GenBank BLASTn search of this sequence revealed 99% similarity with three ITS sequences of E. alphitoides (AB292699, AB292702 and AB292704). Based on the morphological data and ITS sequence analysis, the fungus was identified as E. alphitoides. To our knowledge, this is the first report of E. alphitoides causing powdery mildew on netleaf oak in Mexico.

FIRST REPORT OF CYTOSPORA PUNICAE ASSOCIATED WITH WOOD CANKER AND BRANCH DIEBACK DISEASE OF POMEGRANATE IN TUNISIA

Pomegranate (Punica granatum L.) is an economically important crop in Tunisia with an annual production of 23.000 tons. During May and June of 2014, severe branch dieback was observed in pomegranate plants cv. Gabsi located in Gabes region with about 8% of disease incidence. Symptoms of the disease included leaf yellowing, wood lesion and canker formation. Isolation of the pathogen was performed from 10 samples of active cankers plated onto PDA medium amended with 100 mg l-1 of tetracycline hydrochloride. Fungal colonies were then sub-cultured onto PDA medium at 22°C. All isolates were identified as Cytospora sp. based on colony morphology, conidial characteristics and pycnidia formation (Palavouzis et al., 2015). The isolates developed white mycelium, which turned green to dark brown with hyaline, allantoid, aseptate conidia (average 4-5 μm x 1.75 μm) and production of dark coloured pycnidia 300 to 450 μm in diameter after 15 days (Peduto Hand et al., 2014). Identity of these isolates was confirmed by sequencing the internal transcribed spacer region. The ITS sequences were deposited in GenBank (Accession No. KT272402). These sequences revealed 99% genetic identity with those of Cytospora punicae species available in GenBank (KJ621689; KJ621688). Pathogenicity of Cytospora punicae was evaluated by inoculation of two isolates in 1-year-old shoots of pomegranate cv. Gabsi (Palavouzis et al., 2015). The inoculated shoots developed necrotic spots with vascular discoloration spreading downward and upward from the inoculation site. Cytospora punicae was recovered from 100% of the inoculated shoots. This is the first report of Cytospora punicae causing wood canker and branch dieback of pomegranate in Tunisia.

Streptomyces natalensis programmed cell death and morphological differentiation are dependent on oxidative stress.

Streptomyces are aerobic Gram-positive bacteria characterized by a complex life cycle that includes hyphae differentiation and spore formation. Morphological differentiation is triggered by stressful conditions and takes place in a pro-oxidant environment, which sets the basis for an involvement of the oxidative stress response in this cellular process. Characterization of the phenotypic traits of Streptomyces natalensis ΔkatA1 (mono-functional catalase) and ΔcatR (Fur-like repressor of katA1 expression) strains in solid medium revealed that both mutants had an impaired morphological development process. The sub-lethal oxidative stress caused by the absence of KatA1 resulted in the formation of a highly proliferative and undifferentiated vegetative mycelium, whereas de-repression of CatR regulon, from which KatA1 is the only known representative, resulted in the formation of scarce aerial mycelium. Both mutant strains had the transcription of genes associated with aerial mycelium formation and biosynthesis of the hyphae hydrophobic layer down-regulated. The first round of the programmed cell death (PCD) was inhibited in both strains which caused the prevalence of the transient primary mycelium (MI) over secondary mycelium (MII). Our data shows that the first round of PCD and morphological differentiation in S. natalensis is dependent on oxidative stress in the right amount at the right time.

FIRST REPORT OF WHITE MOULD CAUSED BY SCLEROTINIA SCLEROTIORUM ON MARIGOLD (TAGETES ERECTA) IN BANGLADESH

Marigold (Tagetes erecta) is one of the commercial ornamental flowers commonly grown in Bangladesh. In January 2011, rotten flowers of marigold covered with fluffy white mycelia were found in Rangpur, Bangladesh. Symptoms began from petals and subsequently expanded to the whole flower and the lower part of the plant. Dark brown lesions with necrotic tissues appeared on the infected leaves and stems. Seriously infected plants showed flower dropping and branch wilting. A fungus showing hyaline and septate mycelia was isolated from the infected tissue using potato dextrose agar. The fungus formed round to irregular-shaped sclerotia. Apothecia development from sclerotia were also observed on moist sand. The partial sequence (531 bp, GenBank accession No. AB937102) of the internal transcribed spacer region of rDNA was determined by the method described in Rahman et al. (2015). The result showed a 100% identity with those of Sclerotinia sclerotiorum, which is known as a pathogen to various plants (Purdy, 1979). Pathogenicity of the isolate to marigold was examined by detached leave and flower assays using the mycelial block. After incubation at 20°C with 90% relative humidity in 12 h light/12 h dark conditions, all inoculated leaves and flowers were rotten, while no symptom developed on the controls. The fungus was re-isolated from the symptomatic tissues and Koch’s postulates were confirmed. In Bangladesh, the fungus is becoming an emerging threat to various hosts (e.g. Rahman et al., 2015). To our best knowledge, this is the first report of S. sclerotiorum infection on marigold in the country.