Occurrence of Neopestalotiopsis clavispora Causing Leaf Spot on Dendrobium officinale in China

Dendrobium officinale Kimura L., an endangered orchid plant, is a rare and precious Chinese herb and widely used to prepare Chinese traditional medicine (Zheng et al. 2005). In August 2021, significant indications of an unknown leaf spot disease were observed on greenhouse-grown D. officinale in Yueqing of Wenzhou (28.39°N, 121.04°E), Zhejiang Province, China, the main producing location of this orchid plant. Approximately twenty percent of plants surveyed showed typical infection symptoms. Initially, the symptoms appeared as small, circular black spots. As the disease developed, the center of the lesions was sunken with a black border. To determine the causal agent, 10 symptomatic plant samples were collected and all pieces from symptomatic plant leaves were used for isolating pathogen. Tissues between healthy and necrotic area were cut into pieces (5 × 5 mm, n=10), disinfected with 10% sodium hypochlorite for 1 minute, rinsed 3 times with sterile water, and dried on sterile tissue. Samples were then placed on potato dextrose agar medium (PDA) for 1 piece per plate, and incubated at 25℃ in a dark biochemical incubator. After 3 days, hyphal tips growing from the disinfected tissues were individually transferred to new PDA plates and incubated at 25℃ in the dark. Twelve same fungal isolates were obtained from all symptomatic leave fragments, then DDO11 was chosen as a representative isolate for further study. The colonies showed white aerial mycelium after 5 days culture at 25°C on PDA. Black viscous acervuli appeared and scattered on the surface of the colony after 8-12 days culture. Conidia were spindle shape, five cells, four septa, average 29.3 × 8.5 μm (n = 30; length × width). The apical and basal cells were lighter in color, and most of them were hyaline. The middle three cells were darker in color, and mostly brown. There are 2 to 4 colorless and transparent unbranched accessory filaments at the top, 32.5 µm in average length, and the basal cell has a small appendage, 9.2 µm in average length, n=30. For fungal identification to species level, Internal transcribed spacer (ITS) region, β-tubulin gene (TUB2) and translation elongation factor-1α (TEF-1α) were amplified (Qiu et al. 2020), respectively. The ITS, TUB2 and TEF-1α gene sequences of the representative isolate DDO11 were deposited in NCBI GenBank nucleotide database with accession numbers OK631881, OK655895 and OK655896, respectively. BLASTn analysis respectively showed 100%, 100% and 99.6% nucleotide sequence identity with Neopestalotiopsis clavispora strain accessions MG729690, MG740736 and MH423940, which indicated that the pathogen belonged N. clavispora. A maximum-likelihood phylogenetic analysis based on multi-locus sequence (ITS, TUB2, and TEF-1α) using MEGA X showed the similar result (Kumar et al. 2018). To verify pathogenicity, thirty 1-year-old healthy D. officinale plants of cultivar Yandang1 were used for inoculation tests. Spores of N. clavispora DDO11 were produced on PDA for 7 days at 28°C and washed with sterile distilled water, and the concentrations were adjusted to 1 × 106 spores/ml using a hemocytometer. Fifteen surface disinfected healthy plants were inoculated by spraying the suspension (2 ml, 1 × 106 spores/ml) and covered with plastic bags for 24 h, and another 15 plants treated with sterile distilled water were used as control. The plants were placed in a humidified chamber (>95% relative humidity) at 25°C for 48 h after inoculation and kept in a growth chamber (Kiangnan, China) at 25°C with 12-h day/night cycle for 8 days (Cao et al. 2019). All inoculated leaves showed symptoms identical to those observed in the field. No disease occurred on the controls. The Neopestalotiopsis isolate was reisolated from the symptomatic leaves, and species identification was confirmed by the morphological and molecular method described above. N. clavispora has been reported to cause diseases on a variety of plants all over the world, such as strawberry (Gilardi et al. 2019), blue berry (Shi et al. 2021), Syzygium cumini (Banerjee et al. 2020), Macadamia (Qiu et al. 2020), and so on. To the best of our knowledge, this is the first report of N. clavispora causing leaf spot on D. officinale in China. This report will help us to recognize the leaf spot disease of D. officinale and establish a foundation for future studies on N. clavispora to address effective management strategies.

Efficacy of Aspergillus sp in the Production of Protease Enzyme with Different Substrates

In the present study, the soil samples were collected from marine environment of Arichalmunai, Dhanushkodi, Ramnad District, Tamilnadu ,India. Fungal species were isolated by plating method, in 50% sea water containing potato dextrose agar medium .Totally 16 fungal species were isolated and identified from the soil sample. The production of protease from Aspergillus niger , A.flavus and A. terreus by liquid state fermentation. The production of protease enzyme was optimized by using fermentation medium containing different substrates. The maximum protease production was observed on wheat bran, containing medium. The protease production was maximum in temperature 35ºC were recorded. Wheat bran produced the maximum level protease. The optimization work also carried out. This study revealed that coastal environment provides impressive density of fungi in the East Coast of India and are unexplored for microbial resources can be useful in industry.

Identification and Characterization of Triple Action Bioagents (TAB) and Their Potency against Fusarium Wilt of Lentil

Fusarium wilt is a severe disease that plays a significant role in reducing the yield of lentil. Under favorable conditions for disease growth, the disease can cause complete crop failure and can be a crucial limiting issue for lentil cultivation in specific geographical zones. The current work focused on isolating potentialbio-agents exhibiting copper oxychloride resistance and evaluating their efficacy in seed treatment for ecologically sustainable management of Fusarium wilt of lentil. Seventy biocontrol agent isolates were isolated and tested for resistance by growing them on Potato Dextrose Agar medium (PDA) amended with copper oxychloride at the rate of 2500 ppm. Isolate-H10 and isolate-C9 showed more excellent compatibility with copper oxychloride fungicide with 69 mm and 65 mm radial growths, respectively. The isolates H10 and C9 had the highest inhibitory percentages of 84.30% and 83.94% against Fusarium oxysporum f. sp. lentis, respectively, and the highest phosphorus solubilization index (PSI). Primers (ITS 1 and ITS 4) identified these putative bioagents as Trichoderma harzianum isolate skua-tab-1 and Penicillium crysogenum strain Tab2. Sequences were submitted to the NCBI and assigned the accession numbers MK414603 and MK418066. In pot culture, these isolates also demonstrated their superiority in reducing the disease incidence and severity if seeds were treated with H10 and C9 alone or in combination with copper oxychloride fungicide. The two isolated bioagents exhibit three fundamental properties: compatibility with copper oxychloride, antagonistic activity toward the pathogen fall armyworm, and the ability to dissolve phosphorus minerals.

First Report of Brown Leaf Spot of Rice (Oryza sativa L.) Caused by Bipolaris sorokiniana in Pakistan

Brown leaf spot of rice is one of the major seed-borne diseases and can diminish grain production up to 52% (Barnwal et al. 2013). In 2018, infected leaf samples showing the typical symptoms of brown spots were collected from the vicinity of the University of Agriculture, Faisalabad (31°26'10.3"N 73°03'35.1"E). The symptoms were brown-dark spots, with gray-light gray or brown centers surrounded by dark margins and with chlorotic halos and of oval or cylindrical shapes (5 to 9 mm in diameter). Disease incidence averaged 61% across the seven fields observed. Leaves were collected from the seven infected fields and symptomatic leaf tissues of 5 mm2 were excised from representative necrotic spots in each. These tissues were surface disinfected with 70% ethanol, rinsed with sterile distilled water (SDW), dried by blotting on paper, and placed on potato dextrose agar medium. For pathogen growth, the plates were placed at 25oC (±2oC) with a 12-hour photoperiod for 5 days. Five samples from each of the infected fields were taken for pathogen isolation and among them ten isolates were sub-cultured and purified by using the single spore method. The resulting fungal colonies were fluffy and ranged in color from grayish black/black to light brown. Fifteen conidia were measured that are olivaceous-brown to dark brown in color, elliptical to oblong with narrow (tapered) ends, with 3-10 septa and 35.6-65.4 µm in length x 13.1-25.7 µm in width. Conidiophores were yellowish-brown, geniculate, and solitary (Pratt 2003). For molecular studies, rDNA of the internal transcribed spacer (ITS) region, translation elongation factor (tef), RNA polymerase II second largest subunit (rpb2) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified by using the primers ITS1F/ITS4R (White et al. 1990), EF1-983F/EF1-2218R (Rehner and Buckley 2005), 5F2/7CR (O’Donnell et al. 2007), and GPD1/GPD2 (Berbee et al. 1999) respectively. The sequence of all the amplified gene regions of one SUL-1 isolate was deposited into GenBank with accession numbers MN314844 (ITS), MN326866 (tef), MN990457 (rpb2) and MN990456 (gpd). BLASTn queries of the obtained sequences (ITS, tef, rpb2 and gpd) showed 99-100% homology with the corresponding nucleotide sequences of B. sorokiniana (GenBank accession nos. GU480767, MF490855, LT715652 and MK558818 respectively). To fulfill the Koch’s postulates, twenty rice plants (cv. Basmati-385) were sprayed at 2 to 3 leaf stages by using the two representative isolates with a spore suspension of 105 spores/ml. SDW was sprayed on ten control plants. The plants were covered with polyethylene bags to keep the moisture contents and incubated at 25oC (±2oC) for 7 days. After a week, same symptoms as those described above were observed. In the repeated experiment, B. sorokiniana was re-isolated from the infected rice leaves and confirmed morphologically; fulfill the Koch’s postulates. With grave worry, the other species of the genus Bipolaris (B. oryzae, and B. victoriae) have also been found to the cause brown leaf spot of rice (Motlagh and Kaviani 2008). To our knowledge, this is the first report of Bipolaris sorokiniana causing brown leaf spot of rice in Pakistan. Because rice is highly consumable grain in Pakistan, so the rapid spread of this disease in the rice farming areas is of a serious concern.

Synthesis, Characterization and Biological Evaluation of some Novel Substituted Indole-Coumarin Derivatives as Potential Antibacterial and Antifungal Agents

Aim: In the present work, we have designed and synthesized indole-fused coumarin derivatives as potential antibacterial and antifungal agents. Place and Duration of Study: The present work was carried out at PEA's Modern college of Pharmacy, Sector 21, Yamunanagar, Nigdi- 411044 between the duration of January-2021 to May-2021. Methodology: The in vitro antibacterial and antifungal activity was performed by disc diffusion method. The antibacterial activities were tested using agar nutrient medium against Bacillus subtilis, Bacillus pumilus, Escherichia coli, and Pseudomonas aeruginosa that are representative types of gram-positive and gram-negative organisms respectively. The anti-fungal activity of all the compounds were determined on potato dextrose agar medium against Aspergillus niger and Candida albicans. Clotrimazole 100 μg/ml was used as a standard and DMF was used as control. Results: It was observed that all the compounds were sensitive to the gram +ve bacteria. Compound 2c, 2e, and 2f were sensitive to gram –ve bacteria. Compounds 2a, 2b, and 2f were sensitive to both the fungal strains while all the derivatives were sensitive to Candida albicans. Conclusion: From present investigation it has been observed that compound 2f was sensitive against gram +ve, gram –ve, and both the strains of fungal strains. Structurally, compound 2f possess aldehyde functional group at indole nucleus. Therefore, from present study we have concluded that compound 2f is a lead molecule for the further development of potential antibacterial and antifungal agents.

Effectiveness of fungal, bacterial and yeast antagonists for management of mango anthracnose (Colletotrichum gloeosporioides)

Background Mango anthracnose, caused by Colletotrichum gloeosporioides, is one of the most important diseases of mango crop. It mainly attacks leaves, flowers, young fruits and twigs and also appears as a post-harvest disease of ripened fruits. Application of bio-control agents has huge potential in plant disease management. The goal of the present research was to establish the potential of individual and combined bio-control agents for the management of mango anthracnose under in vitro and under field conditions. Results The antagonistic reaction of six fungi, six bacteria and nine yeasts against C. gloeosporioides on potato dextrose agar medium and malt extract agar medium was observed among which Trichoderma harzianum was found to be the most efficient with 89.26% mycelial growth inhibition. Evaluation of bio-control agents against anthracnose disease development on mango fruit revealed that dip treatment of mango fruits in spore suspension (1.2 × 104 cfu/ml) of T. harzianum for 5 min was the most effective and provided disease control to the tune of 81.67%. Combined application of effective bio-control agents as a post-harvest fruit dip treatment was also evaluated against the mango anthracnose on mango fruits, where the treatment of T. harzianum + Pichia anomala was very effective with 93.39% disease control. Under field conditions, three consecutive sprays of T. harzianum, starting with the initiation of disease on leaves, followed by other two sprays at an interval of 15 days during 2015 and 2016 were found the best for the management of mango anthracnose disease both on leaves and on fruits at two locations. Conclusions The combined and individual applications of bio-control agents, viz.T. harzianum, Bacillus subtilis and P. anomala, through foliar spray or by fruit dip had the potential to control mango anthracnose. The bio-formulations of these bio-control agents had the potential to replace chemical fungicides and also protect the natural environment, thus playing a significant role in integrated disease management.

First Report of Leaf Anthracnose Caused by Colletotrichum karstii of Piper nigrum in Yunnan Province, China

Pepper (Piper nigrum L.) is one of the economically important spice crops of China and mainly grown in the Hainan and Yunnan provinces. In January 2021, the classic anthracnose lesions were observed on pepper leaves at a plantation (24°57’50"N, 98°53’00"E) in Baoshan city, Yunnan, China. Most of the diseased spots occurred at the tips and margins of the old pepper leaves. Lesions were grayish brown or pale white with a slight yellow halo, concentric whorl black dots or scattered black dots were observed on the leaves spots sometimes (Fig. 1). Five symptomatic leaves from different parts of the field were sampled for pathogen isolation. Lesion tissues removed from the border between symptomatic and healthy tissue were surface sterilized in 75% ethanol, then air-dried, plated on potato dextrose agar medium plates (PDA), and incubated in a 12-h photoperiod at 28℃. Similar fungal colonies developed from all plated tissues after 5 days. And five isolates from different leaves (one isolate per leaf) were sub-cultured using the single-spore method. The colonies appeared white, cottony, aerial mycelium dense and slow-growing (mean 1.01 mm day–1) on PDA plates in 6 days. Conidia were short-cylindric, straight, sometimes slightly constricted near the center, ends broadly rounded, measuring 11.05 to 14.43 × 3.78 to 6.08 µm (average = 12.03 × 5.48 µm, n=200). Appressoria were single, subglobose to elliptic, light brown to dark black. Among them, genomic DNA of two isolates (21HJ0301-1 and 21HJ0301-2) were extracted from mycelium and used as a template for molecular identification. The internal transcribed spacer (ITS) region of ribosomal DNA, and partial sequence of chitin synthase (CHS-1), actin (ACT) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene regions were amplified with primer pairs ITS1/ITS4, CHS-79F/CHS-354R, ACT-512F/ACT-783R, GDF/GDR, respectively (Weir et al. 2012). These four gene sequences were deposited in GenBank (Accession No. MZ725047 and MZ725048 for ITS, MZ733415 and MZ733416 for GAPDH, MZ733408 and MZ733409 for ACT, MZ733422 and MZ733423 for CHS-1). A multilocus phylogenetic analysis performed with the reference sequences revealed that both 21HJ0301-1 and 21HJ0301-2 isolates clustered with C. karstii (Fig.2). Based on morphology and molecular results, isolates were confirmed to be C. karstii. Pathogenicity tests were carried out on potted seedlings in the greenhouse, six healthy leaves per isolate were inoculated with six-day-old cultures of C. karstii mycelial discs of 5 mm in diameter after being wounded with a needle or non-wounded. Control leaves were inoculated with PDA agar. Inoculated plants were incubated under high relative humidity at room temperature. Anthracnose symptoms appeared within 5 days using non-wounded or wounded inoculation methods. All control leaves remained asymptomatic. The fungus was re-isolated from inoculated leaves fulfilling Koch’s postulates, but not on controls. C. karstii has a wide range of hosts, such as rubber tree, tea-oil tree, chili, and some other plants belonging to the family Orchidaceae in China (Cai et al. 2016; Jiang and Li 2018; Diao et al. 2017; Yang et al. 2011). To the best of our knowledge, this is the first report of C. karstii on Piper nigrum in China. This report will help us to recognize the anthracnose disease of Piper nigrum and establish a foundation for future studies on C.karstii to address effective management strategies.

ISOLATION OF ENDOPHYTIC FUNGI FROM TRIDAX PROCUMBENS GROWN IN CONTAMINATED SITES AND ASSESSMENT OF THEIR HEAVY METAL TOLERANCE

Seven strains of endophytic fungi were isolated from the leaves of Tridax procumbens and were first screened individually for tolerance to heavy metals like Zn, Pb, Cu, Mg, and Cr from 5 ppm to 50 ppm on Potato dextrose agar medium. They were further tested for resistance at 75, 100, 200, 400, and 600 ppm of heavy metals. The fungal isolate on the PDA medium without any heavy metal was used as a control for growth comparison. It was observed that with increasing concentration of heavy metal from 5 to 600 ppm there was a decrease in the number of fungal isolates. Few of the fungal isolates were able to tolerate heavy metals up to 600 ppm. The screening test revealed heterogeneity in the heavy metal tolerance of the isolates.

Sclerotinia Rot of Peucedanum japonicum Thunb. Caused by Sclerotinia sclerotiorum

Sclerotinia rot was occurred on the leaf and stem of Peucedanum japonicum Thunb. in greenhouse field of Pohang city of Gyeongbuk province in Korea. The typical symptom of the disease was light brown spot and tipburn on infected leaves. The colony of the isolated fungus was white to light gray in color. Asci were cylindrical shape and 75‒240×5.9‒17.3 μm in size. Apothecia were cup-shaped with numerous asci and 0.5‒0.9 cm in size. Ascospores were aseptate and ellipsoid in shape, and 8.4‒10.7×4.8‒5.8 μm in size. Sclerotia formed on the plants and potato dextrose agar medium were globose to irregular in shape and black in color. Partial sequencing of rDNA of this isolate showed that it was 100% consistent with that of Sclerotinia sclerotiorum. It was confirmed that the same lesion was formed by reinoculating this pathogen on a healthy P. japonicum Thunb. and the same strain was isolated. This is the first report on the Sclerotinia rot of P. japonicum Thunb. caused by S. sclerotiorum in Korea.

First Report of Colletotrichum siamense Causing Daylily (Hemerocallis citrina) Anthracnose in China

Daylily (Hemerocallis citrina Baroni) is a perennial herb whose flowers are commonly used in traditional Chinese cuisine. It is commercially cultivated in the Loess plateau of Gansu province, China. From July to October 2020, necrotic lesions were observed on the foliage of daylily plants in Huan County, Gansu, China, with an average disease incidence of 90%, and 52 to 86 disease index across four fields (approximate 6 hectares). Lesions were fusiform or nearly fusiform yellowish-brown spots of different sizes and a yellow irregular border. Older lesions were almost dark brown that often coalesced and expanded to cover the entire leaves. Thirty-four samples were collected from plants with typical foliar symptoms. Symptomatic tissues were excised from the margins of the lesions and sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, rinsed with sterilized water four times, dried on sterile paper towels, and cultured on Potato Dextrose Agar medium at 25°C for 7 days. A total of 34 fungal isolates with 100% isolation frequency were obtained and characterized. Colonies were white, becoming pale brown with age, reverse turned grayish black with age and irregular pale yellowish borders on the reverse side. Conidia (n=50) were hyaline, one-celled, subcylindrical with obtuse to slightly rounded ends, of 12-18.5×3.5-6 µm in size, (avg. 15.5×4.8 µm). The isolates were designated as K2010301 (51-54) and deposited in the Microbiological Culture Collection Center at College of Pastoral Agriculture Science and Technology, Lanzhou University (China). For fungal identification to species level, genomic DNA of a representative isolate (isolate MG) was extracted. Internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase-1 (CHS-1) and beta-tubulin (TUB2) were amplified using V9G/ITS4, GDF1/GDR1, CHS-354R/CHS-79F, and T1/Bt-2b primer sets (Damm et al., 2012), respectively, and deposited in GenBank under accession numbers MW811458, MW836582, MW836581, and MW836584. BLASTn showed higher than 99% identity with Colletotrichum siamense (GenBank: KP703350 (ITS), MN884050 (GAPDH), MN894598 (CHS-1), and KX578815 (TUB2)). A Bayesian inference analysis of the four concatenated loci showed that isolate MG grouped in the C. siamense clade. Pathogenicity tests were performed by spraying a spore suspension (1×105 conidia/mL) of a 10-day-old culture of isolate “MG” onto 3 healthy and asymptomatic daylily plants. Three control plants were only sprayed with the same volume of sterile distilled water. The inoculated plants were covered with black plastic bags for 2 days to maintain high relative humidity. Anthracnose symptoms resembling those observed in the field developed after 7 days on all inoculated plants, while no symptoms were observed on the control plants. The fungus was reisolated and identified as C. siamense based on morphological features and DNA sequence analysis, fulfilling Koch’s postulates. It has been demonstrated that C. liliacearum (Zhuang, 2005), C. gloeosporioides, and C. spaethianum (Yang et al., 2012) are anthracnose pathogens of H. citrina. To our knowledge, this is the first report of C. siamense causing daylily anthracnose worldwide. This fungal pathogen represents a severe threat and has the potential to cause yield losses of daylily, so further studies should focus on epidemiology and effective management strategies of this disease.