Profuse Sporulation Research Articles

Population Structure of Phytophthora infestans in Israel Changes Frequently Due to the Import of Asymptomatic Late Blight-Infected Potato Seed Tubers from Europe.

Late blight, caused by the oomycete Phytophthora infestans, is a devastating disease of potato worldwide. In Israel, potatoes are grown twice a year, in autumn and spring, with late blight causing extensive damage in both seasons. While tuber seeds for the autumn planting are produced locally, seed tubers for the spring planting are imported from Europe due to dormancy of local tubers. Here, we demonstrate that seed tubers imported from Europe for the spring season carry asymptomatic infection with EU genotypes of P. infestans, which alters the population structure of the pathogen each spring. The proportion of imported tubers carrying asymptomatic infections ranged between 1.2 and 3.75%, varying by year and cultivar. Asymptomatic tubers produced late blight-infected sprouts about one month after planting. The sporangia produced on these sprouts served as primary inoculum, causing intensive foliage attacks on neighboring plants. When sprout-infected plants were uprooted and the mother tuber was washed, sliced, and placed in moistened dishes at 18 °C, profuse sporulation of P. infestans developed on the slices' surfaces within 1-2 days. The dominant genotype of P. infestans in the autumn season in Israel is 23A1, but genotypes in the following spring season changed to include 13A2 or 36A2. Surprisingly, genotype 43A1, which might be resistant to CAA and OSBPI fungicides and appeared in Europe in 2022, emerged in Israel in spring 2024. The immigrating genotypes do not persist in the country, allowing 23A1 to regain predominance in the following autumn. Long-term monitoring data suggest that the population structure of P. infestans changes yearly but temporarily due to the import of new genotypes from Europe.

First Report of Alternaria alternata Causing Postharvest Fruit Rot of Indian Jujube (Ziziphus mauritiana) in China.

Ziziphus mauritiana Lam., commonly known as Indian jujube or ber, is a popular fruit crops grown in tropical and sub-tropical regions of China. It is commonly stored at 4℃, relative humidity of about 90%, combined with waxing or sealing with film bag. In January 2023, a postharvest fruit rot was observed on Indian jujube in three markets located in Nanchang city of Jiangxi province, China, with a disease incidence of 4 to 10%. Initially, brown spots appeared on the surface or base of thefruit, which gradually expanded into irregular brown lesions. Gray-white hyphae developed in the center of the lesions, and ultimately thefruitrotted. To isolate the pathogen, small pieces (5 × 5 mm) of ten infected fruits were surface-sterilized in 75% ethanol for 15 s and then 1% sodium hypochlorite for 30 s, rinsed three times in sterile water, plated onto potato dextrose agar (PDA), and incubated at 25°C for 3 days. Eight strains with similar morphological characteristics were isolated, and one representative isolate (JXAA-1) was used for morphological and molecular characterization. The colonies on PDA were initially olive green with white margins, and later turned dark olive or black with profuse sporulation. Conidia were borne singly or in a chain, brown, with 1 to 5 transverse septa and 0 to 3 longitudinal septa, obclavate to obpyriform, and measured 12.9 to 33.7 × 7.5 to 12.9 μm (n = 30). On the basis of morphological characteristics, the isolates were tentatively identified as Alternaria spp. (Simmons 2007). To confirm the identification, genomic DNA was extracted from the isolate JXAA-1 with the Fungi Genomic DNA Extraction Kit (Solarbio Biotech, China). The 18S nrDNA (SSU), 28S nrDNA (LSU), internal transcribed spacer of the rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha (TEF1), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (EndoPG) and an anonymous gene regions (OPA 10-2) were amplified and sequenced using primers NS1/NS4, LR7/LR0R, ITS5/ITS4, gpd1/gpd2, EF1-728F/EF1-986R, Alt-for/Alt-rev, PG3/PG2b, OPA10-2L/OPA10-2R, respectively (Woudenberg et al. 2015). The obtained DNA sequences (SSU: PP190241; LSU: PP190242; ITS: PP189927; GAPDH: PP196557; TEF1: PP196558; Alt a 1: PP196559; EndoPG: PP196560; and OPA 10-2: PP196561) showed 100% homology with those of A. alternata (GenBank accession nos. MT000349 [1020/1020 bp]; KP940477 [1312/1312 bp]; MK972909 [583/583 bp]; MN615421 [593/593 bp]; MN046379 [280/280 bp]; MN304714 [490/490 bp]; MN698284 [458/458 bp] and MH975214 [701/701 bp]). A maximum likelihood phylogenetic tree was constructed by combining all sequenced loci in IQTREE web servers. The isolate JXAA-1 clustered with Alternaria alternata (CBS 121336). The fungus associated with postharvest fruit rot on Z. mauritiana was thus identified as A. alternata. To evaluate the pathogenicity, six surface sterilized fruits were wounded by a sterile scalpel and inoculated with a 10 μl drop of spore suspension (1 × 105 conidia/ml) of isolate JXAA-1. Another six fruits were inoculated with sterilized ddH2O as control and the experiment was repeated three times. All fruits were incubated at 25°C and 80% relative humidity. After 5 days, all the wounded fruit inoculated with A. alternata showed similar symptoms to those observed previously, while the control fruits remained healthy. A. alternata was consistently reisolated from infected fruit and confirmed by morphological and molecular data, fulfilling Koch's postulates. A.alternata has previously been reported causing leaf spot and fruit rot on Chinese jujube (Ziziphus jujuba) in China (Bai et al. 2015; Li et al. 2021). But to our knowledge, this is the first report of A. alternata causing postharvest fruit rot on Indian jujube (Z. mauritiana) in China. Therefore, managers should pay more attention to postharvest fruit rot of jujube caused by A. alternata, the foam bag is put on after the membrane bag is sealed, the broken or infected fruit is picked out in time to reduce the spread of pathogenic fungus.

A Comparative Study of Various Substrate Combinations on Mass Multiplication of Trichoderma lexii (T-94a)

The high cost of raw materials has long hindered the large-scale commercialization of Trichoderma sp. conidiophore formulations. This study addresses the need for affordable, nutrient-rich alternatives by exploring the use of lignocellulosic residues, comprising lignin, cellulose, and hemicellulose, as a carbon source for cultivating Trichoderma. These fungi possess unique attributes, such as filamentous growth and prolific spore production, which make them valuable for composting lignocellulosic waste. Using different substrates with a combination of straws effectively helps in the decomposition of straw and enhances the mass multiplication of Trichoderma lexii (T-94a). Different substrates such as broken rice, grounded maize, gram flour, sorghum & broken wheat were used in combination with paddy straws. A combination of Maize + straw induced an early response in the mycelial growth of Trichoderma lexii (T-94a). After 5 and 10 days of inoculation, physical parameters such as growth and sporulation characteristics of T. lexii (T-94a) show highly uniform growth and profuse sporulation on maize and straw combination as compared to broken rice & straw, sorghum, and straw combinations. This finding has promising implications for improving agricultural practices and waste management. Further research can fine-tune the optimal substrate ratios to enhance the efficiency of biocontrol agents and waste decomposition. Scaling up the application of these substrate combinations in real-world farming scenarios and assessing their long-term environmental impact will be essential for realizing the full potential of these sustainable agricultural practices.

First Report of Trichoderma crassum Causing Leaf Spot on Tomato (Solanum lycopersicum cv. M82) in Ohio.

Trichoderma is a genus of wood-decaying fungi generally found in soil (Druzhinina and Kubicek 2005). Trichoderma crassum was confirmed to be a sister species to T. virens according to the molecular sequencing results (Chaverri et al. 2003). A foliar disease with ~70% incidence on Solanum lycopersicum was observed in a greenhouse at The Ohio State University (40°0'8'' north latitude, 83°1'36'' west longitude), Columbus, United States, in December 2021. On average up to 60% of the leaves per two-month-old tomato plant were infected. Initially, the dark-grey color and irregular spots appeared at the leaf tips. As the disease progressed, the yellow necrotic lesions were observed surrounding the preformed disease spots. Finally, the infected leaves appeared curled and wilted as a whole. The leaf fragments from three tomato plants 40 inches apart were cut from the diseased lesions and surface sterilized with 75% ethanol (30 seconds) and 1% NaOCl (60 seconds), subsequently rinsed with sterilized deionized water three times. Nine pieces of the sterilized leaf tissues were then placed on the PDA plates at 28℃ in the dark and incubated in one incubator for 4 days. The pure cultures of five isolates were acquired and examined with a light microscope. The fungus from all the isolates changed from white to dark green with the radial pattern and profuse sporulation on the PDA. The produced round conidia were observed under a light microscope (Fig S1). The DNA was extracted from two representative isolates which showed the same morphology. The internal transcribed spacer (ITS) region and a conserved fungal rRNA region were amplified using the primers ITS1/ITS4 (5'-TCCGTAGGTGAACCTGCGG-3' and 5'-TCCTCCGCTTATTGATATGC-3') (White et al. 1990) and SR6f/SR7r (5'-TGTTACGACTTTTACTT-3' and 5'-AGTTAAAAAGCTCGTAGTTG-3') (Hirose et al. 2012), respectively. The PCR products were further sequenced by Sanger sequencing (Table S1). Based on the BLAST results through NCBI website, the ITS sequences of the two isolates were 99% (566/572) and 98% (558/572) identical to Trichoderma crassum DAOM 164916 (EU280067). Their SR sequences both showed 99% (290/293; 289/293) identity to the same strain. The phylogenetic tree was also created with the sequences of ITS region by MEGA software (version 11) (Fig S2). Therefore, the fungus was identified as Trichoderma crassum based on its morphological characteristics (green conidia), Sanger sequencing results, and phylogenetic tree. To complete Koch's postulates, the 5-mm-diameter fungal agar discs of 7-day-old pure cultures were used for the inoculation on 18 healthy leaves of six tomato cv. M82 plants with two-month-old. The sterile pure PDA discs of the equal size were used for the mock inoculation as a comparison. Fungal plug method was chosen in this study because it had been widely applied to characterization of the fungal pathogens causing leaf spot disease (Pornsuriya et al. 2020; Yang et al. 2021). Five days later, the same symptom as those that occurred on the previously naturally infected tomato plants were observed on all the inoculated leaves (Fig S3A). However, there were no symptoms on the leaves with the mock inoculation. The fungus re-isolated from the symptomatic leaves showed the consistent morphology (dark-green color with radial sporulation) with the original isolates (Fig S3B). Thus, Trichoderma crassum was verified as the causal agent of the foliar disease on Solanum lycopersicum cv. M82 in our greenhouse. To our knowledge, it is the first report of Trichoderma crassum leading to the leaf spot and wilt on tomato in Ohio. The identification of the causal agent lays the groundwork for the development of necessary disease management techniques. We acknowledge the funding support from CFAES Internal Grants Program 2021009.

First Report of Leaf Spot Disease Caused by Alternaria brassicae on Cercidiphyllum japonicum in China.

Cercidiphyllum japonicum is a deciduous tree belonging to the genus Cercidiphyllum of the family Cercidiphyllaceae (Li et al., 2008). Fossil records indicated that this tree was once distributed throughout the Northern hemisphere during the tertiary period, whereas it is now only found in Japan and China as a consequence of quaternary glaciation. In 1989, C. japonicum was listed as a Rare and Endangered plant in China (Song et al., 1989). It is also highly valued for use in ornamental, medicinal, and research contexts, leading to its widespread planting and cultivation throughout China. In September 2021, a severe leaf spot infection (FigS1.A) was first detected on C. japonicum trees in Meigu County, Sichuan Province, China (N 28°33', E 103°14'). In a survey of twenty 100-year-old C. japonicum trees in this region, the incidence of such leaf spot was found to be approximately 95%. During the early stages of disease, infected leaves exhibited small punctate spots along the leaf center or margins. These spots were brown in the center with black edges. As the disease progressed, these spots expanded until they coalesced to yield large circular or irregularly shaped regions of necrotic tissue, and finally produced mildew. Samples of leaf tissue between symptomatic and healthy regions (5 mm×5 mm) were excised from five symptomatic leaves, surface disinfected for 30 s with 75% ethanol, soaked for 2 min in 3% NaClO, rinsed then plated on potato dextrose agar (PDA) medium supplemented with ampicillin and carbenicillin (50 μg/ml each). After cultured for 3 days in the dark at 25°C, emergent hyphae were purified by subculturing them on fresh PDA medium. In total, single spore culturing was performed by collecting and purifying seven fungal isolates. These isolates exhibited largely comparable morphological characteristics. Aerial hyphae had a cotton-like appearance and were white to pale gray in color (FigS1.B), turning pale reddish-brown with profuse sporulation (FigS1.C). Conidia were present in long chains, with conidiophores being present in clusters or in isolation (FigS1.D), with 1-5 transverse septa, 0-3 oblique and longitudinal septa and an ellipsoidal to obpyriform structure, measuring 9.0-38.6 μm in length and 5.1-12.6 μm in width (n = 40) (FigS1.E). These seven isolates thus exhibited morphological characteristics consistent with those of members of the Alternaria genus (Simmons, 2008). Molecular identification of a representative isolate (LGB9) was performed by amplifying the internal transcribed spacer (ITS) rDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and partial RNA polymerase II largest subunit (RPB2) gene sequences with the ITS1/ITS4 (White et al.,1990), GDF/GDR (Templeton et al., 1992), TEF-728F/TEF-986R (Carbone & Kohn 1999) and RPB2-5F2/RPB2-7cR (Sung et al., 1990; Liu et al., 1999), and Bt-2a/Bt-2b (Glass and Donaldson 1995) primer pairs, respectively. The resultant sequences were deposited in GenBank (ITS, OL659190; GAPDH, OL685343; TEF, ON340848; RPB2, OL685344). Further phylogenetic analyses of isolate LGB9 revealed it to cluster in the A. brassicae clade with 97% bootstrap support. To confirm the pathogenicity of isolate LGB9, 15 healthy leaves from five one-year-old C. japonicum plants were spray-inoculated with a suspension containing 3×105 LGB9 conidia/mL, with control leaves instead being sprayed with distilled water. After 8 days, inoculated leaves exhibited symptoms similar to those observed on naturally infected leaves (FigS1.F-I), whereas the mock leaves were free of any symptoms. This is the first report to our knowledge of a case of leaf spot disease caused by A. brassicae affecting C. japonicum in China or anywhere else in the world. To ensure the protection of this living fossil species, appropriate interventional measures should be adopted to manage the development and spread of this disease.

First Report of Botrytis cinerea Causing Leaf Spot on Strawberry in Florida.

During the 2020-2021 Florida strawberry season (October to April), strawberry (Fragaria × ananassa) plants showing leaf spots were observed on samples submitted to the Diagnostic Clinic at the University of Florida's Gulf Coast Research and Education Center. Disease incidence was up to 5% and observed on four different farms in Plant City, FL on cultivars SensationTM Florida127 and Florida Brilliance. All the samples were submitted early in the season (November) and shared the same nursery source in California. Symptoms consisted of circular or irregular lesions with purple or brown halos, eventually developing leaf blight with sporulation at the center on advanced lesions. Diseased tissues (0.5 mm2) were surface disinfested with 10% bleach solution for 90 s, rinsed twice in sterile deionized water, and plated on general isolation medium (Amiri et al. 2018). Plates were incubated at 25°C and a 12-h photoperiod. A fungus producing white mycelia with sparse sporulation of Botrytis-like spores was consistently isolated. Isolates were single-spored and grown on HA medium to induce sporulation (Leroch et al. 2013). Three isolates (20-291, 20-293, and 20-295) were selected for identification and pathogenicity assays. Resulting cultures on HA had profuse sporulation resembling gray mold. Conidia (n=50) were round to ellipsoid ranging from 9 to 14.6 μm long (Avg=10.8, SD=1.3) and 6.3 to 9.5 μm wide (Avg=7.7, Sd=0.7). No sclerotia formation was observed on GI and HA medium. Based on morphology, the pathogen was tentatively identified as Botrytis cinerea (Hong et al. 2001; Jarvis 1977). DNA was extracted from the same three isolates using the FastDNA kit (MP Biomedicals, Solon, OH), and the heat shock protein (HSP60), RNA polymerase II-binding (RPB2), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) genes were amplified (Staats et al. 2004). Sequences were deposited in GenBank (accession nos. MZ288746 - MZ288754). BLASTn searches revealed that isolates were 100% identical to B. cinerea reported causing leaf spot on strawberry in California; accession numbers MK919494 (HSP60, 996/996 bp), MK919495 (RPB2, 1131/1131 bp), and MK919496 (G3PDH, 877/877 bp). To test for pathogenicity, four one-month-old plants of 'Florida Brilliance' were used per isolate and control treatment. Spores were harvested from two-week-old cultures grown on HA medium, and the suspension adjusted to 106 spores/mL in a solution of 0.1% of Tween 20. Plants were spray inoculated until run-off and kept inside clear plastic boxes for 48 h. Control plants were sprayed with sterile deionized water. Afterward, plants were kept in a misting table in the greenhouse with a water regime of 3 s every 10 min during the day. Disease incidence was evaluated weekly, and the experiment repeated once. Two weeks after inoculation, leaf spots were observed in all inoculated plants, while controls remained healthy. Fungi morphologically identical to the original isolates were re-isolated from the diseased tissues. To our knowledge, this is the first report of B. cinerea causing leaf spot on strawberry in Florida. This disease was recently reported in California (Mansouripour and Holmes 2020), which is where the transplants originated from. Considering the disease was observed early in the Florida season, it is likely that it was introduced with transplants from the nursery. This pathogen is also the causal agent of Botrytis fruit rot, which is considered a major disease of strawberry, and a previous study has shown that populations resistant to multiple fungicides are introduced with transplants (Mertely et al. 2018, Oliveira et al. 2018). While Botrytis leaf spot is currently considered rare and of minor significance (Mansouripour and Holmes 2020), it could contribute to the spread of fungicide resistance to from nursery to strawberry fruit production fields. Efforts should be implemented to monitor its occurrence and spread considering the high variability and fungicide resistance profile of this pathogen.

First Report of Leaf Spot Caused by Alternaria alternata on Yucca gloriosa in China.

Yucca gloriosa L. is introduced to China as a garden plant because of its attractive tubular flowers (Ding et al. 2020). In 2020 and 2021, a foliar disease occurred on approximately 10% of the Y. gloriosa plants in the campus of Henan Institute of Science and Technology, Xinxiang (35°18'N, 113°54'E), Henan Province, China. At the early stages, symptoms appeared as small brown spots on the tip of the leaves. As the disease developed, the spots gradually expanded and turned into necrotic tissue with a clear brown border. The length of lesions ranged from 1 to 3 cm. Infected tissue samples were cut into small pieces, surface sterilized with 75% ethanol for 30 s followed by 0.5% NaClO for 2 min, rinsed thrice with sterile water and plated on potato dextrose agar (PDA). After incubation at 25℃ for 3 days, five fungal isolates were collected and purified using single spore culturing. Morphological observations were made on the 7-day-old cultures. Colonies on PDA were white at first and then turned to dark olive or black along with profuse sporulation. Conidia were borne on branched conidiophores, light brown to dark brown, ellipsoidal to obpyriform, and 20.5 to 43.6 ×7.5 to 15.4 μm in size, with 2-6 transverse septa and 0-3 longitudinal septa (n = 50). The morphological characteristics of the five isolates were consistent with the description for Alternaria alternata (Simmons 2007). One representative isolate (ZQ20) was selected for molecular identification. The internal transcribed spacers (ITS)-rDNA, translation elongation factor-1 alpha (TEF-1α), Alternaria major allergen (Alt a1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene regions were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EFl-728F/ EFI-986R (Carbone and Kohn, 1999), Alt-for/Alt-rev (Hong et al. 2005), and gpd1/gpd2 (Berbee et al. 1999), respectively. Their sequences were submitted to GenBank (ITS, MW832377; TEF-1α, MW848791; Alt a1, MW848792; GAPDH, MW848793). BLAST searches showed ≥99% nucleotide identity to the sequences of A. alternata (ITS, 100% to KF465761; TEF-1α, 100% to MT133312; Alt a1, 100% to KY923227; and GAPDH, 99% to MK683863). Thus, the fungus was identified as A. alternata based on its morphological and molecular characteristics. To confirm its pathogenicity, 25 healthy leaves of five 2-year-old Y. gloriosa plants were used. Leaves were wounded with one sterile needle and inoculated with 5-mm-diameter fungal agar disks obtained from 5-day-old cultures. Sterile PDA disks of the same size were used as the controls. Treated plants were covered with a plastic bag at 12 to 25℃ for 48 h to ensure a high level of moisture. After 15 days, the inoculated plants developed the symptoms similar to those observed in naturally infected plants, whereas the control plants were symptomless. The fungus was reisolated from the symptomatic leaves with the same morphological and molecular characteristics as the original isolates, fulfilling the Koch's postulates. Leaf spot caused by A. alternata in the Yucca plants has been reported in India (Pandey 2019). To our knowledge, this is the first report of A. alternata causing leaf spot on Y. gloriosa in China. Identification of the cause of the disease is important to developing effective disease management strategies.

First Report of Alternaria alternata Causing Leaf Spot of Tartary Buckwheat (Fagopyrum tataricum) in China.

Buckwheat (Fagopyrum tataricum) is recognized as a healthy food with abundant nutrients and high levels of rutin. In April and May of 2020, an unknown tartary buckwheat leaf spot distinct from Nigrospora leaf spot (Shen et al. 2020) was observed in Xiangxiang, Hunan, China (27°49'54″N, 112°span style="font-family:'Times New Roman'; color:#0000ff">18'48″E.). Disease incidence was 60-70% within three fields (totally 7, 000 m2). The disease occurred after plants emerged. Initial symptoms began as circular, or ellipsoid, chlorotic, water-soaked spots, mostly on leaf apexes or leaf margins. The small spots gradually enlarged and often coalesced to form large circular or irregular, pale to light brown lesions, and the infected leaves eventually withered and fell off. Thirty 2 × 2 mm infected tissue pieces collected from five locations were sterilized in 70% ethanol for 10 S, in 2% NaClO for 30 S, rinsed in sterile water for three times, dried, and placed on PDA with lactic acid (3 ml/L). After 3-5 days at 28°C in the dark, 17 fungal isolates were purified using single-spore isolation method. Almost all fungal isolates had similar morphology. Colonies were initially olive green with white margin and later turned dark olive or black with profuse sporulation. Conidia were borne in long chains, tawny to brownish green, with 1-3 longitudinal and 1-7 transverse septa, pyriform, and measured 9.5-39.6 µm long, and 5.1-12.6 µm wide (n=50). Based on morphological characteristics, the fungus was identified as Alternaria alternata (Simmons 2007). Partial internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α(TEF) and Alternaria major allergen (Alt a1) genes of isolate BLS-1 were amplified using ITS1/ITS4 (Mills et al. 1992), EF1-728F/EF1-986R (Carbone and Kohn 1999), Gpd1/Gpd2 and Alt-4for/Alt-4rev (Lawrence et al. 2013), respectively. Sequences were deposited into GenBank with acc. nos MW453091 (ITS), MW480219 (GAPDH), MW480218 (TEF), and MW480220 (Alt a1). BLASTn analysis showed 99.8% (ITS, MH854758.1), 100% (GAPDH, KP124155.1), 99.8% (TEF, KP125073.1) and 100% (Alt a1, KP123847.1) identity with reference strain CBS 106.24 of A. alternata, confirming isolate BSL-1 to be A. alternata. A neighbor-joining phylogenetic tree constructed by MEGA7.0 based on concatenated sequences of the four genes indicated that BSL-1 formed a distinct clade with A. alternata CBS 106.24 with 100% bootstrap values. Pathogenicity test was triplicately performed on healthy leaves. Twenty leaves of five 20-day-old plants (cv. Pinku1) were sprayed with conidial suspension (1×106 conidia/ml) collected from PDA cultures with 0.05% Tween 20. An equal number of control leaves were sprayed with sterile water to serve as the controls. Treated plants were kept in a greenhouse at 28±3 °C with relative humidity of 80±5% for 24 h and transferred to natural conditions (22-30°C, RH 50-60%). After 4 to 6 days, all inoculated leaves developed symptoms similar to those observed in the fields, while the control leaves remained healthy. A. alternata was re-isolated from all infected leaves. Occasionally-isolated Diaporthe isolates were not pathogenic. A. alternata causes leaf spot of oat (Zhao et al. 2020) and leaf blight of F. esculentum (Lu et al. 2019). To our knowledge, this is the first report of A. alternata causing leaf spot on F. tataricum in China and the world. Effective strategies should be developed to manage the disease.

The hop downy mildew pathogen Pseudoperonospora humuli.

Pseudoperonospora humuli is an obligate biotrophic oomycete that causes downy mildew, one of the most devastating diseases of cultivated hop, Humulus lupulus. Downy mildew occurs in all production areas of the crop in the Northern Hemisphere and Argentina. The pathogen overwinters in hop crowns and roots, and causes considerable crop loss. Downy mildew is managed by sanitation practices, planting of resistant cultivars, and fungicide applications. However, the scarcity of sources of host resistance and fungicide resistance in pathogen populations complicates disease management. This review summarizes the current knowledge on the symptoms of the disease, life cycle, virulence factors, and management of hop downy mildew, including various forecasting systems available in the world. Additionally, recent developments in genomics and effector discovery, and the future prospects of using such resources in successful disease management are also discussed.TaxonomyClass: Oomycota; Order: Peronosporales; Family: Peronosporaceae; Genus: Pseudoperonospora; Species: Pseudoperonospora humuli.Disease symptomsThe disease is characterized by systemically infected chlorotic shoots called “spikes". Leaf symptoms and signs include angular chlorotic lesions and profuse sporulation on the abaxial side of the leaf. Under severe disease pressure, dark brown discolouration or lesions are observed on cones. Infected crowns have brown to black streaks when cut open. Cultivars highly susceptible to crown rot may die at this phase of the disease cycle without producing shoots. However, foliar symptoms may not be present on plants with systemically infected root systems.Infection processPathogen mycelium overwinters in buds and crowns, and emerges on infected shoots in spring. Profuse sporulation occurs on infected tissues and sporangia are released and dispersed by air currents. Under favourable conditions, sporangia germinate and produce biflagellate zoospores that infect healthy tissue, thus perpetuating the infection cycle. Though oospores are produced in infected tissues, their role in the infection cycle is not defined.ControlDowny mildew on hop is managed by a combination of sanitation practices and timely fungicide applications. Forecasting systems are used to time fungicide applications for successful management of the disease.Useful Websites https://content.ces.ncsu.edu/hop‐downy‐mildew (North Carolina State University disease factsheet), https://www.canr.msu.edu/resources/michigan‐hop‐management‐guide (Michigan Hop Management Guide), http://uspest.org/risk/models (Oregon State University Integrated Plant Protection Center degree‐day model for hop downy mildew), https://www.usahops.org/cabinet/data/Field‐Guide.pdf (Field Guide for Integrated Pest Management in Hops).

First Report of Leaf Spot Caused by Alternaria alternata on Sonchus asper in China.

Sonchus asper (Spiny sowthistle), belonging to the Asteraceae, is a problematic weed in grain crops, orchards as well as turf (Cho et al., 2019). In April 2016, about 90% of the S. asper plants infesting a pear orchard had symptomatic with black spots densely distributed on the leaves and stems in Da Yang Town, Luyang District, Hefei, Anhui (N31°55'43″, E117°11'40″). The foliar lesions were mostly circular (3.0 mm to 1.5 cm in diameter) or irregular in shape; lesions on the stem were elliptical to irregular in shape running along the vascular bundle and about 3 to 6mm long × 2 to 3mm wide. Severely affected plants had their leaves become completely blighted and eventually the plants died. Fifteen tissue pieces from five symptomatic leaves and stems were surface sterilized with 0.5% NaClO for two min, rinsed three times with sterile water, and then plated on 1/4-strength potato dextrose agar (PDA) medium. Fourteen fungal isolated were obtained from the tissues, the isolation frequency from the plant tissue pieces was almost 93%. Fungal colonies were circular, initially white, and eventually turned to dark olive or black along with profuse sporulation. The conidia were tawny to brownish green, obclavate to obpyriform, with a cylindrical or coniform short beak, ranging from 16.8 to 39.8 μm long × 6.3 to 14.7 μm wide with two to five transverse and zero to two longitudinal septa (n = 50) and were borne on branched conidiophores. These morphological characteristics were similar to those described for Alternaria alternata (Simmons, 2007). For one representative isolate, the internal transcribed spacer (ITS) region of rDNA, partial coding sequence of the actin (ACT) gene, partial Alt a 1 major allergen (ALT) gene and partial Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene were amplified and sequenced with primers ITS1/ITS4 (White et al., 1990), ACT512F/ACT783R (Carbone and Kohn, 1999), Alt-4for/Alt-4rev and gpd1/gpd2 (Lawrence et al., 2013) respectively. Sequences were submitted to GenBank (ITS: MH886523; ACT: MH892480; ALT: MN655781; GAPDH: MN655782). Based on a BLAST analysis, ITS, ACT gene and GAPDH gene had 100%-99.8% identity with the existing sequences of the ex-type CBS 916.96 of A. alternata (Fries) Keissler (ITS: AF347031; ACT: JQ671702; GAPDH: AY278808, respectively) and ALT gene showed 100% identity with the A. alternata isolate SCWC10 (MG199093). Thus the pathogen was identified as A. alternata based on its morphological and molecular characteristics. Pathogenicity tests were conducted on six to seven-leaf stage potted S. asper plants (one plant per pot). Five mm diameter fungal disks, which were excised from the edge of a three-day old A. alternata colony, were placed on healthy leaves of five plants. Same size sterile PDA disks were used as controls. In addition, a conidial suspension (107 conidia/ml) was also inoculated on healthy leaves of five plants, sterile distilled water was used as control. Both inoculation experiments were conducted in the greenhouse at 25 ± 2°C, 90 % relative humidity, 12 h light/dark photoperiod and repeated at least three times. Lesions were observed on all the leaves within 12 h after fungal disk inoculation. All pathogen inoculated plants developed lesions, similar to those observed on the symptomatic leaves in the field 12 days after conidial inoculation. No symptoms were observed on both control treatments. A. alternata was re-isolated from the inoculated leaves and confirmed by both morphological and molecular techniques, confirming Koch's postulates. To our knowledge, this is the first report of A. alternata causing leaf spot on S. asper in China. The pathogen could cause severe disease in S. asper, and has the potential to be further studied as a fungal weed biocontrol.

Phenotypic characterization in natural population of Spot Blotch (Bipolaris sorokiniana) of Barley (Hordeum vulgare L.)

A study was undertaken on phenotypic characterization of different isolates of Bipolaris sorokiniana isolated from the different varietal trials and nurseries of barley at the agricultural research farm, Institute of Agricultural sciences, Banaras Hindu University. The isolation was performed by transfer of single spore of the pathogen from diseased leaf in Potato Dextrose Agar media (PDA) under aseptic condition. The experiment revealed five groups of isolates on the basis of colony colour, morphology and number of spores. It was found that (42.2%) of the isolates were black in colour with profuse sporulation and suppressed type growth, (35.71%) of the isolates were greyish in colour and (21.42%) of the isolates possessed whitish colony with full plate growth and lower sporulation. The size of conidia ranged from 24 × 13µm to 36 × 14µm in size, while the number of spores per mm2 of colony varied from 20.36 to 162.77.

Effect of incubation temperatures on mycelial growth, conidial features and density of Stemphylium botryosum Walr isolates

The cultural and morphological variability of Stemphylium botryosum Walr was studied with five isolates viz. isolate 1 (Stb-I1), isolate 2 (Stb-I2), isolate 3 (Stb- I3), isolate 4 (Stb-I4) and isolate 5 (Stb-I5) collected from Parsa, Rautahat, Dang, Banke and Chitwan districts of Nepal respectively during 2011-2012 crop season with Completely Randomized Design (CRD) maintaining 3 replications at Plant Pathology Laboratory of Grain Legumes Research Program (GLRP), Rampur, Chitwan. Single spore isolation technique was followed to isolate the pathogen. The pathogen was grow on Potato Dextrose Agar (PDA) and incubated with different temperatures in BOD incubator. The calibrated ocular micrometer was used to measure the length and breadth of the conidia while conidial count with sporulation intensity was attempted with hemocytometer. The mycelium color of the different isolates was varied from white, grey, brown and brownish black in peripheral part while mostly black pigmentation was noticed in lateral part of the culture plate. The shape and texture was regular and velvety for most isolates and in some isolate, roughly irregular shape with cottony texture was found. The higher colony diameter of 7.66 cm and conidial dimension of 29.42×18.12 μm (L/B ratio-1.62) with profuse sporulation intensity (46.67 conidia /0.01 ml) was observed at 25 °C after 15 days of incubation on PDA. However, 2.84 cm diameter, 19.58×9.81 μm (L/B ratio-2.00) with poor conidial density (7.60 conidia /0.01 ml) was noticed at 10 °C. The observation at 10 and 35 °C was at par in case of mycelial growth, conidial features and sporulation intensity. Mostly conidia of all isolates were brown in color at lower incubation temperature but they became dark grey brown at 25 and 30 °C and grey color was noticed when incubated at 35 °C on PDA after 15 days of incubation.Kathmandu University Journal of Science, Engineering and TechnologyVol. 12, No. I, June, 2016, page: 80-89

How Does Phytophthora infestans Evade Control Efforts? Modern Insight Into the Late Blight Disease.

The infamous oomycete Phytophthora infestans has been a persistent threat to potato and tomato production worldwide, causing the diseases known as late blight. This pathogen has proved to be remarkably adept at overcoming control strategies including host-based resistance and fungicides. This review describes the features of P. infestans that make it such a daunting challenge to agriculture. These include a stealthy lifestyle that helps P. infestans evade plant defenses, effectors that suppress host defenses and promote susceptibility, profuse sporulation with a short latent period that enables rapid dissemination, and a genome structure that promotes the adaptive evolution of P. infestans by fostering genetic diversity. Nevertheless, there is reason to be optimistic that accumulated knowledge about the biology of P. infestans and its hosts will lead to improved management of late blight.

In vitro Sporulation, Cultural Characterization, Identification and Phylogeny of Gray Mold Fungus [Botryotinia ricini (Godfrey) Whetzel.)] of Castor (Ricinus communis L.)

Control of Botryotinia ricini (Godfrey) Whetzel., gray mold pathogen of castor, is a challenge due to the lack of known sources of complete resistance and effective disease management practices. Isolation of pure culture of causal pathogen and its profuse sporulation are imperative for any pathological or molecular understanding of infection or resistance mechanisms and host-pathogen interactions. Season-dependent disease occurrence coupled with sparse conidial production in vitro has been a serious constraint. A culture medium (BRS) based on V8 juice broth supplemented with salts and minerals was developed and culture method and conditions optimized for profuse in vitro sporulation of gray mold fungus. ITS barcoding identified the isolate from Hyderabad as Amphobotyrs ricini (A. ricini isolate IRHT-S1) which was closely related to A. ricini strain Cop-Ar5. Phylogenetic analysis of the isolate with 20 related fungal species revealed that A. ricini forms a separate clade. Studies on growth response, culture characteristics and sporulation of A. ricini in different media ingredients revealed that the culture morphology differed with medium composition, especially in medium with 60 g L−1 dextrose, while capsule/epicarp extract was dispensable. The addition of salts such as MgSO4 (1.0 g L1), KCl (1.0 g L−1) and FeSO4.7H2O (0.02 g L−1) favoured sporulation.

Efficacy of fosetyl-Al, propamocarb, dimethomorph, cymoxanil, metalaxyl and metalaxyl-M in Czech Pseudoperonospora cubensis populations during the years 2005 through 2010

A total of 134 Czech Pseudoperonospora cubensis isolates originating from Cucumis sativus were used for fungicide resistance screening. Efficacy of six commonly used and registered fungicides was screened in Czech P. cubensis populations. The investigations covered the frequency and occurrence of sensitive, moderately resistant and resistant isolates to the individual fungicides during the period 2005–2010. Fosetyl-Al (Aliette 80 W) and propamocarb (Previcur 607 SL) were the most effective fungicides. All tested isolates were sensitive on all tested fosetyl-Al concentrations. However, some isolates expressed resistance (profuse sporulation) or moderate resistance (limited sporulation) to lower and/or even to recommended concentrations of propamocarb in the years 2006 and 2008–2010. Metalaxyl (Ridomil PLUS 48 WP) and metalaxyl-M (Ridomil Gold MZ 68 WP) were ineffective. Isolates collected in 2008 and 2009 showed large variation in moderate resistance or resistance even at high fungicide dosages. However, this result was not confirmed in 2010, when efficacy of these fungicides increased slightly, and 69% and 43% of isolates were controlled at the recommended concentration of metalaxyl-M and metalaxyl, respectively. Limited or profuse sporulation was observed only sporadically at high concentrations. Sensitivity of isolates to cymoxanil (Curzate K) differed among the studied years. While cymoxanil was ineffective in the years 2005–2008 and in 2010, 68% of isolates were controlled at the recommended concentration in 2009. During the years 2005 through 2010, a shift towards higher sensitivity to dimethomorph was observed at all screened concentrations.

Aeroponic Propagation of VAMycorrhizal Spores for Soil Inoculation as a Biofertilizer

Onion (Allium cepa) and Barley (Hordeum vulgare) colonized by Glomus mosseae, G. fasiculatum, and G. intraradices were grown in aeroponic cultures. After 14 weeks, all roots were colonized by the inoculated vesicular-arbuscular mycorrhizal fungi. Abundant vesicles and arbuscules formed in the roots, and profuse sporulation was detected intra- and extra-radically. Within each fungal species, barley contained significantly more roots and spores per plant than onion did, although the percent root colonization was similar for both hosts. Mean percent root colonization and sporulation per centimetre of colonized root generally increased with time, although with some treatments colonization declined by week 14. Spore production ranged from 4 spores per cm of colonized root for G. fasiculatum to 51 spores per cm for G. intraradices. Infectivity trials with root inocula resulted in a mean ranged from 28.9 to 51.1% of onion roots colonized byG. mosseae, G. fasiculatum, and G. intraradices, respectively, while in aeroponic culture it ranged from 33.8 to 38. However, infectivity studies comparing G. fasiculatum spores from soil and aeroponic culture indicated no biological differences between the spore sources. Aeroponically produced G. mosseae and G. fasiculatum inocula retained their infectivity after cold storage (4°C) in either sterile water or moist vermiculite for at least 4 and 9 months, respectively.

First Report of Phakopsora pachyrhizi Adapting to Soybean Genotypes with Rpp1 or Rpp6 Rust Resistance Genes in Field Plots in the United States.

During the years following the first detection of soybean rust, caused by Phakopsora pachyrhizi Syd., in the continental United States in November, 2004, soybean (Glycine max [L.] Merr.) genotypes with the Rpp1 or Rpp6 resistance genes exhibited high levels of resistance there (1,2,3). When challenged with 72 different American isolates collected between 2006 and 2009, PI 200492 (source of Rpp1) produced no sporulating lesions (2). In 2011 and 2012, however, field populations of P. pachyrhizi from Gadsden County, FL, caused higher rust severity on plants with Rpp1 or Rpp6 than in previous years. To assess aggressiveness, sporulation ratings were made using a 1 to 5 scale (no sporulation to profuse sporulation) on leaflets collected from field plants at or near the R6 (full seed) stage of development. A dissecting microscope was used to examine 3 replications of 5 leaflets each in 2009 or 2 replications of 10 leaflets each in 2012. The sporulation ratings increased on PI 200492 (from 1.1 ± 0.1 in 2009 to 4.1 ± 0.4 in 2012), PI 567102B (Rpp6; from 1.1 ± 0.1 in 2009 to 2.4 ± 0.2 in 2012), and L85-2378, a 'Williams 82' isoline carrying the Rpp1 gene (from 1.0 ± 0 in 2009 to 4.0 ± 0.3 in 2012). The mean 2009 and 2012 sporulation ratings for susceptible control Williams 82 were 5.0 ± 0 and 4.2 ± 0.1, respectively. Single-uredinium-derived isolates were purified from bulk isolates collected from field plots in 2009 (FL-Q09-1), 2011 (FL-Q11-1), and 2012 (FL-Q12-1). Greenhouse and detached leaflet assays were then used to test the virulence of these isolates under controlled conditions. Detached leaflets from 3-week-old seedlings of Williams 82, PI 200492, PI 567102B, and L85-2378 were inoculated by pipetting 15-μl drops of a 30 to 40 urediniospore μl-1 suspension onto the abaxial side of 3 to 4 leaflets per genotype, which were then sealed in Petri plates and incubated in a growth chamber at 20 to 22°C. Plates were kept in the dark for 12 h following inoculation. For the greenhouse assay, the first trifoliolate leaves of at least 3 seedlings were each sprayed with 1.5 ml of a 40 urediniospore μl-1 suspension and incubated 24 h at 22 to 24°C in a dark mist chamber. The plants were then maintained at 22 to 24°C and 76 to 86% relative humidity in a greenhouse with 10 h of daylight on average. Two weeks after inoculation with FL-Q11-1 or FL-Q12-1, all of the genotypes had developed TAN lesions with abundant sporulation, indicating susceptibility. On leaves inoculated with FL-Q09-1, however, no visible reaction was observed on PI 200492, and PI 567102B developed reddish-brown (RB) lesions associated with incomplete resistance. Although the lesions on Rpp1 and Rpp6 greenhouse seedlings inoculated with the FL-Q11-1 and FL-Q12-1 isolates were slightly darker than those that developed on Williams 82 plants or on detached leaflets, the profuse sporulation that is characteristic of the TAN infection type was observed. The higher virulence of the 2011 and 2012 Florida isolates on two soybean genotypes with Rpp1 and one with Rpp6 confirmed the presence of a P. pachyrhizi pathotype in north-central Florida that is more virulent against these genes than earlier populations from the southeastern United States.

Development of mutants of Melanocarpus albomyces for hyperproduction of xylanase

The wild type filamentous fungus, Melanocarpus albomyces, produces many commercially valuable enzymes, including Xylanases and Xylan-debranching enzymes with low activity. In this paper, we report for the first time the development of M. albomyces mutants from vegetative spores. Profuse sporulation of M. albomyces was induced on Potato Carrot Agar medium. These spores, when subjected to chemical mutation, led to the isolation of the hyper-xylanase producing mutant, viz, M. albomyces IITD3A. Various parameters including number of spores, nitrogen source and C/N ratio of the medium were optimized for production of xylanase by the mutant in a shake flask culture. Under controlled pH at 7.8, the mutant produced highly active xylanase with 415 IU/mL after 36 h of growth on soluble alkaline lignocellulosic extract in a 14-L fermentor. The overall productivity of xylanase was 8-fold higher than the wild type culture with11, 530 IU/L/h. The enzyme can be easily stored at 37°C for 50 days by addition of a small amount of the preservative — thiomersal. Also, for long term storage, a lyophilized powder form of the enzyme can be used which retained 100% of its activity for > 50 days. When assayed at pH 7.5 and temperature 55°C, the xylanase retained 100% of its original activity, and also at pH 9.0, it retained > 50% of its activity for 2 h, which is promising for its application in the pulp and paper industry.

Characterization of Kudzu (Pueraria spp.) Resistance to Phakopsora pachyrhizi, the Causal Agent of Soybean Rust

Kudzu (Pueraria spp.) is an accessory host for soybean rust (SBR) (caused by Phakopsora pachyrhizi) that is widespread throughout the southeastern United States. An expanded survey of kudzu sites was conducted in 2008 to determine the proportion of natural resistance in the north-Florida kudzu population. Of the 139 sites evaluated, approximately 18% were found to be free of SBR infection, while 23% had reduced sporulation. Ten accessions of kudzu from north-central Florida were characterized for their response to challenge by a single isolate of P. pachyrhizi under laboratory conditions. Three outcomes were observed: tan lesions with profuse sporulation (susceptible); reddish-brown lesions with delayed, reduced sporulation (resistant); and an immune response in which no lesions developed (immune). Of the 10 accessions, 6 were susceptible, 3 were immune, and 1 was resistant. Cytological examination revealed that resistant interactions were typified by early onset of a multicell hypersensitive response (HR) while typical immune interactions were the result of cell wall depositions that blocked penetration in combination with early onset of the HR. Quantitative real-time polymerase chain reaction was performed to determine the extent of colonization. After 15 days, there was 10-fold less P. pachyrhizi DNA present in resistant compared with susceptible kudzu, while the amount of P. pachyrhizi DNA present in the immune kudzu was below the detection level. Susceptible kudzu had approximately half the amount of P. pachyrhizi DNA present when compared with a susceptible soybean cultivar.

Natural Occurrence of Phytophthora infestans Causing Late Blight on Woody Nightshade (Solanum dulcamara) in New York.

Woody nightshade (Solanum dulcamara) is a common hedgerow herbaceous perennial in the United States, one of only three native Solanum spp. S. dulcamara is a known host of Phytophthora infestans (3), but infection is rarely reported. There is a U.S. record from Maryland (2); in 1947, Peterson (4) stated that this species had never been found blighted in its natural habitat, although in 1960 it was listed as a host of P. infestans in New York (1). The A2 mating type has not been reported on this host. On 2 July, 2009, leaf lesions similar to those of P. infestans on potato were found on wild S. dulcamara at Riverhead, NY. The plant was growing in a home garden within 10 m of potato and tomato plants infected with P. infestans. When two infected leaves of S. dulcamara were incubated for 24 h under high humidity, a pathogen growth developed around the lesion margins that was characterized by hyaline mycelium bearing lemon-shaped sporangia that released motile zoospores after chilling in water, which is consistent with P. infestans. The caducous and limoniform to ovoid sporangia were 39 to 50 μm (average 45 μm) × 26 to 28 μm (average 27 μm) with a length/breadth ratio of 1.66. No oospores were observed. Three isolates were obtained from this plant during July 2009. Growth on rye agar was indistinguishable from that of local tomato isolates of P. infestans. Detached leaflets of S. dulcamara and S. tuberosum, inoculated with the woody nightshade isolates and kept in a humid chamber, became infected and developed profuse sporulation within 5 days. The pathogen isolated was confirmed as P. infestans by morphological, biochemical, and molecular characteristics. Inoculations of attached leaves of potted S. dulcamara plants resulted in necrotic lesions with many sporangia; sporulation also developed on inoculated, attached, and detached tomato leaves. P. infestans was reisolated and identity confirmed as before. The three isolates were A2 mating type, metalaxyl-resistant, mitochondrial haplotype Ia. All were glucose-6-phosphate isomerase 100/122 and peptidase 100/100, as confirmed with single-spore isolates. RG57 fingerprint analysis confirmed that isolates from woody nightshade, tomato, and potato obtained from the same and nearby sites were identical. Although P. infestans in the United States belongs to the new population, which may infect a wider host range than the old US-1 clonal lineage, S. dulcamara infections have only been found when late blight is already widespread in neighboring fields and there is no evidence to suggest that woody nightshade acts as an overwintering host in the United States.