The first detection of Botryosphaeria sinensis on white lupine

Fungal strain MF KP-12.1, which is similar in morphological and cultural characteristics to Botryosphaeria fungi, was isolated from the seeds of white lupine variety Dega grown in Oryol region in 2018. Phylogenetic analysis of the large rRNA subunit (LSU), elongation factor-1α (TEF) and internal transcribed spacer (ITS) fragments was used to accurately identify the isolated strain as B. sinensis. When 2-week-old white lupine plants were treated under laboratory conditions with mycelial suspension of B. sinensis MF KP-12.1, plant damage reached 67 % on day 3, and 100 % plant death was observed on day 10. Pathogenicity of B. sinensis MF KP-12.1 was confirmed by re-isolation of the strain from damaged plant tissue. This is the first detection of B. sinensis on white lupine, which has not been previously reported as a host for this fungus. It is also the first detection of B. sinensis in Russia.

Development and Application of an in Vitro Method to Evaluate Anthracnose Resistance in Soybean Germplasm

Background: Anthracnose caused by Colletotrichum truncatum is a major fungal disease of soybean, especially vegetable soybean (edamame). Studies of this disease have mainly focused on resistance evaluation, but the primary methods used—in vivo inoculation of pods or plants under greenhouse or field conditions—have limitations with respect to accuracy, stability, scale, and environmental safety. Result: In this study, we developed a method for inoculating pods in vitro that entails soaking in a mycelial suspension. We optimized the crucial components, including the mycelial suspension concentration (40 to 60 mg mL-1), maturity of sampled pods (15 days after flowering), and post-inoculation incubation period (5 days). Application of the mycelial suspension by spraying rather than soaking improved the efficiency of inoculation and made large-scale evaluation possible. Using this method, we evaluated 589 soybean germplasm resources [275 cultivars (C), 233 landraces (L), and 81 wild-type accessions (W)]. This screening resulted 37 highly resistant (25 C, 11 L, and 1 W), 148 resistant (100 C, 43 L, and 5 W), 210 moderately susceptible (116 C, 77 L, and 17 W), 91 susceptible (23 C, 52 L, and 16 W), and 103 highly susceptible (11 C, 50 L, and 42 W) materials. Conclusions: The resistance levels of soybean germplasm resources were effectively distinguished by the method we developed. We thus identified many resistant cultivars but only a few resistant landraces and wild-type germplasm resources. Our results will greatly aid future research on soybean anthracnose resistance, including gene discovery, elucidation of molecular mechanisms, and the breeding of resistant cultivars.

Standardization of inoculation techniques for sheath blight of rice caused by Rhizoctonia solani (Kuhn)

To develop a simple and reliable inoculation technique using inoculum sources viz., mycelial suspension, mycelial ball, sclerotia and soil inoculation with homogenized mycelia suspension was carried out. The efficiency of different inoculation techniques were tested on susceptible rice variety Rejendra Sweta. Sheath inoculation with sclerotia gave lesion length 10.33 and 12.33 cm after seven and 15 days of inoculation, respectively. It also shows more significance in terms of relative lesion height to plant height i.e. 30.06 % followed by soil inoculation with homogenized mycelial suspension i.e. 21.62 %. However, relative number of lesions as compared to control was found to be maximum (429.18%) in mycelial suspension spray, followed by soil inoculation using homogenized mycelia suspension (400.43%). The disease rating of sheath blight of rice was also found to be maximum (5) in sheath inoculation with sclerotia followed by soil inoculation of mycelial suspension (3). The disease rating was minimum (1) in sheath inoculation with mycelial ball and foliar spray with mycelial suspension.

Screening Progenies of Mexican Race Avocado Genotypes for Resistance to Phytophthora cinnamomi Rands

Because of the low availability of avocado rootstocks with resistance to Phytophthora cinnamomi, it is necessary to search for genotypes that offer resistance and that could be used as commercial rootstocks. The objective of this study was to select progeny from the genotypes of Mexican race avocado plants that are resistant to P. cinnamomi. Seedlings from 12 avocado genotypes were placed in containers inoculated with a mycelial suspension of P. cinnamomi. Signs of disease in the upper part of the seedlings were registered every 3 days for 8 weeks using a visual scale of damage severity. The χ2 test (P < 0.009) showed significant differences among the genotypes evaluated, with ‘Todo el Año’ being the most resistant, as demonstrated by its rating of 70% asymptomatic seedlings, followed by ‘Plátano’ with 40%. The most susceptible genotypes were ‘María Elena’, ‘Silvestre’, and ‘Hass’, with 100% mortality. Seedling inoculation facilitated the detection of resistance to P. cinnamomi. ‘Todo el Año’ showed resistance toward P. cinnamomi. Therefore, individuals of its offspring could be recommended for use as rootstocks after confirming their resistance with a second evaluation, as well as performing tests in multiple localities to demonstrate their productive behavior after grafting.

Antimicrobial activity of Agaricus brasiliensis on Plasmopara viticola and its effect on the induction of resistance to the control of downy mildew on ‘Isabel Precoce’

ABSTRACT: Agaricus brasiliensis include bioactive compounds that can act as antibiotics, bacteriostatic, fungistatic and nematostatic substances. In this sense, this study aimed to evaluate the effect of a single application of aqueous mycelial suspension (AMS) of A. brasiliensis in control of downy mildew (Plasmopara viticola) and resistance induction in ‘Isabel Precoce’ grapevines under greenhouse conditions. Treatments consisted of three doses of 1%, 5%, 10%, 15% and 20% AMS A. brasiliensis, as well as treatment with acibenzolar-S-methyl (ASM). The variables analyzed were: sporangiospore germination, disease severity, represented by the area under the disease progress curve (AUDPC), catalase enzyme activity, peroxidase and polyphenol. The 10%, 15% and 20% doses of AMS caused approximately 80% reduction in germination of P. viticola sporangiospores. The treatments did not show significant effects in reducing both the AUDPC of mildew and polyphenol oxidase enzyme activity. The A. brasiliensis aqueous mycelial suspension showed a fungitoxic effect on the germination of sporangiopores; however, it was not enough to reduce the severity of mildew in the ‘Isabel Precoce’ grapevines, even when acting on the catalase and peroxidase enzymes. Thus, experiments should be performed to verify the viability of the reproductive structures of the pathogen externalized in the vines when treated with A. brasiliensis AMS.

First Report of Septoria apocyni Causing Spot Blight on the Species of Apocynum venetum and Poacynum pictum in China

The species of Apocynum venetum and Poacynum pictum grow widely from the middle to northwestern regions of China. During the summers of 2011 to 2013, a spot blight was found in wild and cultivated both species in Altay Prefecture of the Xinjiang Uygur Autonomous Region, China. The spot blight caused leaf yellowing and leaf drop, and serious damage to plant phloem. Lesions were circular to irregular, and the diameter of lesions on A. venetum and P. pictum was 1.84 to 6.84 × 1.23 to 4.24 mm and 2.05 to 7.09 × 1.46 to 5.65 mm, respectively. Pycnidia were 70 to 115 × 52 to 120 μm, scattered, spherical, buried, and had a brown hard shell with a prominent ostiole. Conidia were colorless, needle-shaped, or linear. The conidia base was obtuse, containing 3 to 5 indistinct septa, 46.3 to 110.3 × 2 to 2.5 μm. Fungal cultures were obtained by cutting 1-cm-long infected leaf pieces from the margins of the lesions following routine surface sterilizing procedures. The sections were placed on potato dextrose agar (PDA) in petri dishes and incubated at 23°C for 4 weeks (4). Hyphae had septa, the aerial and base mycelium was white and rufous, and the back of the colony was sunken and cracked after 2 weeks, but no spore was observed. To verify the identity, total DNA was extracted directly from fungal mycelium with a UNIQ-10 fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China) and PCR amplification performed with primers ITS1/ITS4 (3). A 512-bp PCR product was sequenced and contrasted with GenBank sequences using BLAST, which revealed 99% identity with Septoria sp. (GenBank Accession No. KC134322.1). To confirm pathogenicity, A. venetum and P. pictum were planted in pots and grown in a greenhouse. After 6 weeks of growth, plants were inoculated by spraying a mycelial suspension onto the foliage while control plants received a similar application of sterilized distilled water. Five pots (3 plants per pot) were used for each treatment. The pots were then placed on plates filled with tap water and covered with Plexiglas hoods in the greenhouse at 20 to 25°C. Lesions began to appear 6 to 7 days after inoculation with the mycelial suspension, whereas control plants remained healthy. The average disease incidence was 19.3%. The symptoms and morphology were similar to Septoria apocyni in Teterevnikova (2). It was determined that spot blight of A. venetum and P. pictum was caused by S. apocyni based on morphological comparison. There is one relevant literature report of spot blight on A. venetum and P. pictum in China, but without any details of the pathogenicity or morphology of the pathogen (1). We believe that this is the first report of S. apocyni occurring on the species of A. venetum and P. pictum in China. References: (1) W. Sun et al. Special Economic Animal and Plant 8:23, 2005. (2) D. N. Teterevnikova. Page 79 in: Septoria sp. Fungus of USSR. Armenian Academy of Sciences Publishing, Armenia, USSR, 1987. (3) G. J. M. Verkley et al. Mycologia 96:558, 2004. (4) W. Zhang et al. Plant Dis. 96:1374, 2012.

First Report of Neofusicoccum ribis Causing Postharvest Decay of Apple Fruit from Cold Storage in Pennsylvania

Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.), previously known as Botryosphaeria ribis (Grossenb. & Duggar), is an aggressive fungal plant pathogen that is part of the N. ribis/N. parvum species complex that causes stem cankers on a variety of woody plant species (2). An isolate of N. ribis was obtained from decayed ‘Honeycrisp’ apple fruit from a commercial cold storage facility located in Pennsylvania in October of 2011. The decayed apple fruit sample had a brownish lesion that was soft, dry, and leathery on the surface while sporulation was not evident. To conduct Koch's postulates, three ‘Golden Delicious’ apple fruits were wound-inoculated with a 50-μl mycelial suspension, obtained from aseptically scraping a 7-day-old potato dextrose agar (PDA) culture of the fungus, and was repeated using ‘Fuji’ apple fruit. The inoculated fruit developed lesions, while water-inoculated fruit were symptomless after 5 days at 20°C. N. ribis was reisolated from infected tissue and was morphologically identical to the original isolate. Genomic DNA was isolated, a portion of the β-tubulin gene was amplified with the gene specific primers, and the amplicon was sequenced and analyzed using BLAST (1). The nucleotide sequence (GenBank Accession No. KC47853) had 99% identity with N. ribis SEGA8 isolate (JN607146.1). The N. ribis isolate produced a grayish-white mycelium with abundant aerial hyphae on PDA and had an olive-colored reverse. Microscopic investigation revealed septate mycelia with right angle branching and conidiomata were not evident on PDA, V8, oatmeal agar (OMA), or water agar (WA). Growth on WA was sparse and transparent, and aerial mycelial growth was not produced. Growth rate analyses were conducted on PDA, V8, and OMA and were 10.1 (±1.39), 20.4 (±1.15), and 17.6 (±0.70) mm/day at 20°C and the experiment was repeated. The minimum inhibitory concentrations (MIC) for the N. ribis isolate was carried out for three postharvest fungicides as described by Pianzzola et al. (3). Briefly, 96 well plates were filled with PDA alone (0 ppm) and PDA amended with 10 fungicide concentrations ranging from 1 to 1,200 ppm for thiabendazole (Mertect), and 1 to 1,000 ppm for fludioxonil (Scholar) and pyrimethanil (Penbotec). A mycelial suspension of the fungus was obtained from pure culture, 50 μl of the mycelial suspension was pipetted into each well, and allowed to grow for 72 h at 25°C. The experiment was conducted twice. The N. ribis isolate displayed MIC values of >1 ppm thiabendazole (Mertect), >1 ppm fludioxonil (Scholar), and 50 ppm pyrimethanil (Penbotec), which are all well below the labeled application rates for these postharvest fungicides. To our knowledge, this is the first report of N. ribis causing postharvest decay on apple fruit obtained from a commercial storage facility in Pennsylvania. References: (1) S. F. Altschul et al. J. Mol. Biol. 215:403, 1990. (2) D. Pavlic et al. Mycologia 101:636, 2009. (3) M. J. Pianzzola et al. Plant Dis. 88:23, 2004.

First Report of Botryosphaeria dothidea Causing White Rot on Apple Fruit in Maryland

Botryosphaeria dothidea (Moug.:Fr.) Ces. De Not. causes perennial cankers on apple trees and causes white rot on apple fruit in the field and during storage (1). Prolonged periods of warm wet weather favor rapid disease outbreaks that result in severe losses, which range from 25 to 50% for the southeastern United States (3). A B. dothidea isolate was obtained from decayed ‘Fuji’ apple fruit exhibiting white rot symptoms from a local farm market in Beltsville, MD, in May 2010. The fruit had characteristic large dark brown lesions with irregular margins and decay expanded unevenly toward the core and the tissue was soft. The pathogen was isolated from symptomatic tissue by spraying the lesion surface with 70% ethanol. The skin with aseptically removed with a scalpel and small pieces of tissue were placed on potato dextrose agar (PDA) and incubated at 20°C. Once fungal growth was evident, the cultures were hyphal-tip transferred to individual PDA plates and incubated at 20°C. The B. dothidea isolate produced black aerial mycelium with a white margin on PDA and had a black reverse. Conidiomata were evident after 10 to 14 days at 20°C only on oatmeal agar. Conidia were hyaline, smooth and straight, fusiform with an subobtuse apex and a truncate base 20 to 26 (24.33) × 4 to 7 (5) μm (n = 50). Genomic DNA was isolated from the fungus and amplified with gene specific primers (ITS 4 and 5) for the ribosomal DNA internal transcribed spacer region ITSI-5.8S-ITS2 as described by White et al. (4). Both forward and reverse strands of the 542-bp amplicon were sequenced and assembled into a contig. The nucleotide sequence (GenBank Accession No. KC473852) indicated 99% identity to B. dothidea isolate CMM3938 (JX513645.1) and to voucher specimens CMW 25686, 25696, and 25222 (FM955381.1, FM955379.1, and FM955377,1). Koch's postulates were conducted using three ‘Golden Delicious’ apple fruit that were wound-inoculated with 50 μl of a mycelial suspension of the fungus, obtained from aseptically scraping a 7-day-old PDA culture, and was also repeated using ‘Fuji’ apple fruit. Large, brown, slightly sunken, soft lesions with undefined edges developed 5 days after inoculation at 20°C and water-only inoculated fruit were symptomless. The fungus was reisolated from infected tissue and was morphologically identical to the original isolate from decayed apple fruit. To determine if the B. dothidea isolate was resistant to postharvest fungicides, the minimum inhibitory concentration (MIC) was conducted using the 96 well plate method with a mycelial suspension of the fungus as described by Pianzzola et al. (2). The MIC for the isolate was >1 ppm for Mertect and Scholar and 50 ppm for Penbotec, which are well below the labeled rates for these postharvest fungicides and the experiment was repeated. To our knowledge, this is the first report of B. dothidea causing white rot on apple fruit in Maryland. References: (1) A. R. Biggs and S. S. Miller. HortScience 38:400, 2003. (2) M. J. Pianzzola et al. Plant Dis. 88:23, 2004. (3) T. B. Sutton. White rot and black rot. Pages 16-20 in: Compendium of Apple and Pear Diseases, A. L. Jones and H. S. Aldwinckle, eds. The American Phytopathological Society, St Paul, MN, 1991. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Application. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

First Report of Nothofagus macrocarpa Dieback Caused by Phytophthora citrophthora and P. nicotianae in Chile

The genus Nothofagus, family Nothofagaceae, comprises 36 species of trees that are native to the Southern Hemisphere. N. macrocarpa (DC.) F.M. Vásquez & R.A. Rodríguez (Roble de Santiago) is an important deciduous tree, endemic to central Chile (32 to 35°S), and found above 800 m altitude. There is an increasing interest in N. macrocarpa as an ornamental. However, a general dieback (40 to 50% prevalence) was observed at a commercial nursery in Santiago in 2009, limiting its multiplication. Symptoms are wilting, partial defoliation, reddish brown cankers on the crowns, and root necrosis. The purpose of this work was to study the etiology of the dieback in nurseries. Phytophthora was isolated from the roots and cankers of symptomatic plants (n = 3) and soil samples (using apples and avocados as baits) on amended corn meal agar (3) at 20°C for 5 days in the dark. Morphologically, P. citrophthora (Smith & Smith) Leonian, and P. nicotianae Breda de Haan were identified (2). On V8 juice agar (V8) (1), P. citrophthora formed petaloid colonies, grew between 5 and 30°C (optimum of 25°C), and produced deciduous, mono- or bipapillated sporangia of (28.1) 45.0 to 64.1 × (18.8) 32.0 to 39.2 μm. On V8, P. nicotianae produced cottony colonies, grew between 10 and 30°C (optimum of 25°C), and produced spherical, intercalary chlamydospores (mean diameter of 19.6 μm) and persistent, papillate, spherical to ovoid, ellipsoid, obpyriform sporangia of (33.2) 47.5 to 67.6 × (24.1) 30.0 to 48.9 μm. Isolates of P. citrophthora were sexually sterile, but P. nicotianae formed oogonia with amphigenous antheridia in dual cultures with P. cinnamomi (A2 compatibility type). BLAST analysis of the internal transcribed spacer (ITS) region of rDNA of isolates identified as P. citrophthora (IMI 399056 and IMI 399054, GenBank Accession Nos. JF699756 and JF699755) and P. nicotianae (IMI 399055, Accession No. JF699757), amplified by PCR using ITS universal primers (4), revealed 100% similarity with reference isolates of P. citrophthora (Accession Nos. GU259324.1 and GU259317.1) and P. nicotianae (Accession No. GU983635.1). P. citrophthora (n = 2) and P. nicotianae (n = 1) were pathogenic when wounded detached twigs (n = 5) of N. macrocarpa and N. obliqua were inoculated with 20 μl of a mycelial suspension (106 CFU/ml) of either Phytophthora spp. Twigs were placed in a moist chamber at 20°C for 12 days prior to determine the length of the necrotic lesions that developed. An equal number of noninoculated twigs were left as control. Reisolation of P. citrophthora and P. nicotianae from inoculated material was 100%. The length of the necrotic lesions (13 to 80 mm) from inoculated N. macrocarpa and N. obliqua was significantly greater (P < 0.05) compared with the controls. Regardless of Phytophthora isolates, necrotic lesions (53.9 ± 15.8 mm) in infected N. macrocarpa were significantly longer than in N. obliqua (28.6 ± 13.1 mm) (P < 0.0001). To our knowledge, this is the first report of P. citrophthora and P. nicotianae associated with dieback on N. macrocarpa in Chile. Therefore, there is a potential risk of Phytophthora dieback in N. macrocarpa in nature. References: (1) J. Ampuero et al. Plant Dis. 92:1529, 2008. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) B. A. Latorre and R. Muñoz. Plant Dis. 77:715, 1993. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Leaf Spot Caused by Phoma multirostrata on Fuchsia × hybrida in Italy

Fuchsia × hybrida (Onagraceae) is widely used in gardens and very much appreciated as a potted plant. During the summer of 2008, a severe foliar disease was observed on 1- to 2-year-old plants in several gardens located near Biella (northern Italy). Small necrotic spots were observed on the upper and lower sides of infected leaves. Spots enlarged to form round areas of 2 to 12 mm in diameter and were well defined by a brown-purple margin at temperatures between 15 and 25°C. Severely infected leaves wilted and abscised as disease progressed. The disease occurred on 100% of the plants and at least 30% of the leaf surface was affected. Stems and flowers were not affected by the disease. A fungus was consistently isolated from infected leaves on potato dextrose agar amended with 25 mg/liter of streptomycin. The fungus was grown on leaf extract agar, including 30 g of autoclaved fuchsia leaves per liter, and maintained at 22°C (12-h light, 12-h dark). After 30 days, black pycnidia 150 to 450 μm in diameter developed, releasing abundant hyaline, elliptical, nonseptate conidia measuring 5.6 to 14.3 (10.3) × 1.9 to 5.6 (3.5) μm. On the basis of these morphological characteristics, the fungus was identified as a Phoma sp. (2). The internal transcribed spacer (ITS) region of rDNA of the isolate coded FuHy1 was amplified using primers ITS4/ITS6 (3) and sequenced. BLAST analysis (1) of the 488-bp segment obtained showed an E-value of 0.0 with Phoma multirostrata. The nucleotide sequence has been assigned GenBank Accession No. GU220539. Pathogenicity tests were performed by spraying leaves of healthy 6-month-old potted Fuchsia × hybrida plants with a spore and mycelial suspension (1 × 106 spores or mycelial fragments per milliliter). Noninoculated plants sprayed with water served as controls. Five plants were used for each treatment. Plants were covered with plastic bags for 5 days after inoculation and kept under greenhouse conditions at 20 to 24°C. Symptoms previously described developed on leaves 12 days after inoculation, whereas control plants remained healthy. The fungus was consistently reisolated from the lesions of the inoculated plants. The pathogenicity test was carried out twice. To our knowledge, this is the first report of the presence of P. multirostrata on fuchsia in Italy as well as worldwide. The importance of the disease is still limited in Italy. References: (1) S. F. Altschud et al. Nucleic Acids Res. 25:3389, 1997. (2) G. H. Boerema and G. J. Bollen. Persoonia 8:111, 1975. (3) D. E. L. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997.