Morphological and Molecular Analysis of Fungal Species Associated with Blast and Brown Spot Diseases of Oryzae sativa

Fungal diseases; blast, and brown spot in rice incur severe yield losses worldwide. Blast is caused by Magnaporthe oryzae, while Bipolaris oryzae is reported as the main causal organism of brown spot. Both diseases cause leaf lesions which are difficult to be differentiated by symptomatology until the late stages. Early detection and differentiation of the lesions would help the adoption of disease management strategies specific to the pathogen and will prevent the native impact on the quality and quantity of rice yields. This study was conducted in the Northern Province of Sri Lanka over five consecutive rice cultivating seasons to characterize the causal fungi of rice blast and brown spot diseases by morphological and molecular means and to develop a visual guide to differentiate the two diseases. Disease incidence was recorded in 114 fields from 2017 to 2019, and fungal isolates associated with lesions of both the diseases were cultured and subjected to morphological and molecular characterization. Competitive growth interaction between M. oryzae and the more common individual fungal isolates of the brown spot lesions, was evaluated. Fungal metagenomics analysis was conducted for the fungal spp. isolated from brown spot lesions. A suppression of blast accompanied by an increased incidence of brown spot disease was observed during the study period. M. oryzae was confirmed to be the causal organism of the blast while over 20 species of fungi were identified to be associated with brown spot lesions through morphological, molecular studies, and metagenomics analyses. Fungal ITS region sequencing revealed considerable genetic variation in the highly conserved region of DNA sequences of blast and brown spot fungal isolates. B. oryzae, Curvularia, and Microdochium species were commonly isolated from brown spot lesions. In vitro competitive growth interaction among the fungal isolates revealed growth suppression of M. oryzae by the fungal isolates associated with the brown spot lesions. Similarly, it can be speculated that the abundance and severity of blast in the field may have an influence on brown spot associated fungi. A simple visual guide was developed to differentiate blast and brown spot lesions. The findings would be highly useful in the timely management of these major fungal diseases affecting rice.

First Report of Southern Blight Caused by Sclerotium delphinii on Euonymus fortunei in China

Euonymus fortunei is an evergreen shrub-vine in the family Celastraceae, widely used as a groundcover or a vine to climb walls, or traditional herbal medicine in China. In August 2019, typical southern blight symptoms that included basal stem rot and the presence of sclerotia in rotted tissue were observed on E. fortunei in Kunshan city, Jiangsu province, China. Disease incidence was estimated at approximately 15 to 20%; meanwhile, approximately 30 to 40% of diseased plants died. The infected plants showed brown to dark stem necrosis near the base, leaf yellowing and wilting. White mycelia and white to dark reddish-brown sclerotia were observed at the base of the stem and rotten tissue. To isolate the causal organism, infected stem tissue and sclerotia collected from diseased plants in a median strip in Kunshan (31°23'40"N, 120°54'57"E) were disinfected with 70% ethanol for 2 to 3 sec, followed by 2 min in 5% NaClO, rinsed three times with sterile water, then plated on potato dextrose agar (PDA) medium, and incubated at 25°C. Isolated colonies were subcultured by needle tip transfer 3 days later. Isolates had white mycelia on PDA, with a radial growth rate of 15.2 to 18.7 mm/day. White and orange sclerotia were developed after 5 to 8 days and eventually turned dark reddish-brown. The sclerotia were globoid or irregular with surface markings (1.4 to 4.3 mm diam.; mean = 2.59 mm; n = 50) on PDA, and the average number of sclerotia produced per Petri dish ranged from 35 to 85 (mean = 52; n = 10). Microscopic observations found septal hyphae and clamp connections. These morphological features were identical to the description of Sclerotium delphinii (syn. Sclerotium rolfsii var. delphinii) (Mukherjee et al. 2015; Punja and Damiani 1996; Stevens 1931). A representative isolate YKY2020.01 was stored in the Key laboratory of National Forestry and Grassland Administration on Ecological Landscaping of challenging Urban Sites in Shanghai. For molecular identification, DNA of the isolate YKY2020.01 was extracted using the Fungal DNA Kit (OMEGA bio-tek, China). The internal transcribed spacer region (ITS fragment including ITS1, 5.8S rDNA, and ITS2 region) was amplified with primers ITS1/ITS4 (White et al. 1990), and then sequenced by Sangon Biotech (Shanghai, China). BLAST analysis in NCBI found the ITS sequence of YKY2020.01 (MW916955) was 99.84% similar to S. delphinii strain CBS272.30 (MH855140). Phylogenetic analysis using maximum likelihood (ML) method placed isolate YKY2020.01 in the same clade as S. delphinii. To evaluate pathogenicity, hyphal blocks (0.7 cm diam.) were placed at the base of the stem of healthy E. fortunei (n = 5 plants). Five healthy plants were inoculated by uncolonized agar blocks as controls. All plants were kept in a greenhouse with a temperature range from 21 to 25.6°C (mean = 24.9°C) and relative humidity of 50%. Inoculated plants were symptomatic after 3 days and wilted after 12 days. Symptoms in inoculated plants were similar to those observed under natural conditions, whereas the control group remained asymptomatic. The fungal pathogen was reisolated from symptomatic tissue and confirmed as S. delphinii. To the best of our knowledge, this is the first report of S. delphinii causing southern blight on E. fortunei in China and worldwide. This finding provides concise and practical information on the newly emerged disease of E. fortunei, which is beneficial for future disease management. References: Mukherjee, A. K., et al. 2015. J. Plant Pathol. 97:303. Punja, Z. K. and Damiani, A. 1996. Mycologia 88:694. Stevens, F. L. 1931. Mycologia 23:204. White, T. J., et al. 1990. Page 315 in PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Funding: This work was supported by the Key Project of Science and Technology Commission of Shanghai Municipality (19DZ1204102).

Evaluation of nutritional amendments and fungicides against brown leaf spot of rice

Helminthosporium oryzae, the causal organism of bacterial leaf spot (BLS) has brought a tremendous loss of rice crop in the field. Rice seedlings of four commercial varieties (Nayab basmati, Kiran 434, Super basmati, and Pak 386) were screened against BLS in the field by using a randomized complete block design (RCBD). The present experiment was encompassed the evaluation of fermented farmyard manure, leaf manure, Boron, and NPK solution against BLS. The effect of different fungicides (Score, Flumax, Melodyduo, and Polyram) was also checked on the mycelial growth of Helminthosporium oryzae in-vitro and in-vivo. The results showed that Pak 386 was highly resistant with 17.22% disease severity and Super Basmati was found moderately resistant with 22.31% disease severity; variety Kisan 434 was found moderately susceptible (24.15%) and Nayab Basmati (24.82%) was susceptible. Among all NPK solutions showed the best results in growth enhancement, plant vigor as well as reduction in disease severity followed by FFYM, boron and leaf manure. Under in-vitro conditions, all the fungicides performed best at 150 ppm in terms of colony growth reduction. The maximum growth inhibition was recorded in Score followed by Flumax, Melodyduo, and Polyram. Score gave a significant reduction in BLS disease severity and maximized the agronomic attributes. The fungicides performed best at 150 ppm in terms of colony growth reduction. The maximum growth inhibition was recorded in Score followed by Flumax, Melodyduo, and Polyram. Score gave a significant reduction in BLS disease severity and maximized the agronomic attributes. Keywords: HBV, HCV, ICT, PCR, Risk factors, Mansehra

Neofusicoccum batangarum Causing Dieback of Mango (Mangifera indica) in Florida

Mango (Mangifera indica) is an economically significant crop, and is affected by dieback in nearly all commercial production areas. Due to the wide range of organisms previously associated with these disease symptoms in Florida, isolations and pathogenicity tests were carried out to determine the causal organism. The pathogen was identified as Neofusicoccum batangarum based on genetic sequences from three loci (internal transcribed spacer of the rDNA (ITS), β-tubulin (BT), and translation elongation factor 1-α (EF)), recommended for members of the Botryosphaeriaceae family. Possible infection routes were determined by inoculating wounded and unwounded stems with N. batangarum. Trees wounded prior to pathogen inoculation developed larger lesions (5.85 cm ± 1.51) than unwounded trees (0.51 cm ± 0.48), p < 0.0003. In addition, lesions only developed at a small number of inoculation sites in the absence of wounds (14.3%), compared to 93% when stems were wounded. No necrosis was observed in the negative controls. This study provides molecular data on N. batangarum, and evidence of its role causing mango dieback in Florida.

Screening Tomato Genotypes for Bacterial Wilt Disease (Ralstonia solanacearum) Resistance in Ghana

Thirteen-tomato genotypes were assembled from the USA, Taiwan and Ghana for screening for bacterial wilt resistance in two environments: in a screen house at Crops Research Institute, Kumasi, Ghana and in the field at a bacterial wilt hot spot at Bechem in the Brong-Ahafo region of Ghana. The bacterium, Ralstonia solanacearum was isolated using the serial dilution method on Nutrient Agar (NA) and aseptically cultured for inoculation. The bacterium was implicated as the causal organism of the bacterial wilt disease of tomato. A local check, Petofake and H7996 were used as susceptible and resistant checks respectively. Three genotypes, H7996, LA0442 and LA0443 demonstrated significant resistance stability in both experimental environments by recording significantly low wilt incidence and severity. H7996 was obtained from the World Vegetable Centre (WVC), Taiwan while the other two genotypes were collected from the University of California, Davis, USA. Apart from the highest yielding genotype LA 0442 that recorded 50.67 t/h, the rest recorded very low yields. The lowest yielding genotype AVTO 1713, recorded (12.67 t/h). Thus, the highest yielding genotype, LA 0442 over-yielded the lowest AVTO1713 by approximately 300%. It is instructive to note that LA0442, which was identified as a stable resistant genotype also recorded the highest yield. This result supports the potential of developing a resistant, high yielding variety using LA0442 as a parent. The future of the tomato industry in Ghana therefore looks promising.

Stagonosporopsis pogostemonis: A Novel Ascomycete Fungus Causing Leaf Spot and Stem Blight on Pogostemon cablin (Lamiaceae) in South China

Pogostemon cablin is one of the well-known Southern Chinese medicinal plants with detoxification, anti-bacterial, anti-inflammatory, and other pharmacological functions. Identification and characterization of phytopathogens on P. cablin are of great significance for the prevention and control of diseases. From spring to summer of 2019 and 2020, a leaf spot disease on Pogostemon cablin was observed in Guangdong Province, South China. The pathogen was isolated and identified based on both morphological and DNA molecular approaches. The molecular identification was conducted using multi-gene sequence analysis of large subunit (LSU), the nuclear ribosomal internal transcribed spacer (ITS), beta-tubulin (β-tubulin), and RNA polymerase II (rpb2) genes. The causal organism was identified as Stagonosporopsis pogostemonis, a novel fungal species. Pathogenicity of Stagonosporopsis pogostemonis on P. cablin was fulfilled via confining the Koch's postulates, causing leaf spots and stem blight disease. This is the first report of leaf spot diseases on P. cablin caused by Stagonosporopsis species worldwide.

Management of Anthracnose Disease of Aloe vera in Bangladesh

Aims: To identify the causal organisms of anthracnose disease of Aloe vera in Bangladesh and to manage this disease in field condition. Study Design: The experiment was designed by Randomized Complete Block Design (RCBD) with three replications. Place and Duration of Study: The field experiments were conducted in Natore, Bangladesh and the laboratory experiments were carried out at the Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh from January, 2017 to December, 2018. Methodology: The causal organism Colletotrichum gloeosporioides was isolated by tissue plating method and identified based on morphological and cultural characteristics and that was confirmed by pathogenicity test. Infested farmer’s fields were selected in rainy season under natural epiphytic condition to evaluate the efficacy of eleven treatments. Results: Among the treatments, Bordeaux mixture gave best result against this disease. Moreover, Tilt 250 EC and Folicur 25 EC and Garlic bulb extract showed better effect against the disease than the other treatments. Lime also has moderate effect against anthracnose disease of A. vera. In 2017, after 4th spray, the lowest incidence was recorded in Bordeaux mixture (58.33%) which was statistically identical with Folicur (64.58%), Tilt (64.58%) and Garlic bulb extract (66.67%). Similarly, the lowest disease severity was found in Bordeaux mixture (3.55) followed by Folicur (5.67%), Tilt (6.67%) and Garlic bulb extract (7.67%). Similar result also found in 2018. After 4th spray, the lowest incidence was recorded in Bordeaux mixture (38.58%) which was statistically identical with Lime (41.66%) and Garlic bulb extract (45.83%). Similarly, the lowest disease severity was found in Bordeaux mixture (0.20%) followed by Lime (0.25%) and Garlic bulb extract (0.36%). Conclusion: Garlic bulb extract could be used as eco-friendly approach. Moreover, use of Bordeaux mixture is better than the traditional use of lime. From chemical pesticides, Tilt 250 EC and Folicur 25 EC could be used for controlling the disease as the last option.

First Report of Epicoccum nigrum Causing Brown Leaf Spot in Tea in Guizhou Province, China

Brown leaf spots were observed on tea [Camellia sinensis (L.) Kuntze] in Sinan County (27.74 °N, 108.35 °E) and Kaiyang County (27.96 °N, 107.34 °E), Guizhou Province, China, from 2018 to 2020. For the leaf spots with the typical symptoms, the disease incidence was estimated to range between 56% and 61%, respectively. The disease severity was estimated to range from 39 to 43 across 12 tea plantations, respectively. The disease initially occurred at the margins of leaf tips, and the lesions expanded gradually, being dark brown and irregularly shaped and became necrotic. To identify the causal organism, two leaves from each of 15 tea twigs, one or two per plantation, were detached from 8- or 10-year-old tea plants on each of 12 plantations. Samples taken from the lesion margins were sterilized with 75% ethanol followed by 0.5% NaOCl, placed on potato dextrose agar (PDA), and then incubated at 25oC in darkness for 5 days (Wang et al. 2020). For each sample, hyphal tips from the margin of a growing colony were successively transferred to fresh PDA, and pure cultures were obtained. Three representative strains were grown on PDA, malt extract agar (MEA), and oatmeal agar (OA) plates. The colonies had smooth margins and abundant mycelia on all three media, with the colony colors being from gray to light purple on PDA, white on MEA, and purplish-red on OA at 5 days post-inoculation. At 20 days post-inoculation on MEA, stromata began to gradually form, which were droplet-like, 100 to 2,000 μm in diameter, and semi-immersed on the medium’s surface. Black sporodochia were produced on the surfaces of stromata. Conidiophores were aggregated in sporodochia, densely compacted, and dark brown. Conidia were globose or pyriform, dark, multicellular, and measured 22.95 ± 3.59 × 19.82 ± 3.13 μm (n = 50) in diameter. The morphological characteristics of the mycelia and reproductive structures of the strains were identical to those of Epicoccum nigrum. The internal transcribed spacer (ITS) region of rDNA, and the partial 28S large subunit rDNA (LSU), RNA polymerase II second largest subunit (RPB2), and beta-tubulin (TUB) genes of these strains were amplified using the primers V9G/ITS4 (De Hoog and Gerrits van den Ende 1998; White et al. 1990), LR0R/LR5 (Rehner and Samuels 1994), RPB2-5F2/fRPB2-7cR (Sung et al. 2007), and TUB2Fd/TUB4Rd (Woudenberg et al. 2009), respectively, and deposited in GenBank (accession no. MW646378, MW291537, MW602293, and MW602295 for ITS, LSU, RBP2, and TUB, respectively). A maximum parsimony phylogenetic analysis indicated that the representative strains clustered with E. nigrum CBS 173.73 (Chen et al. 2017). Pathogenicity tests were performed on 5-year-old potted tea and on 10-year-old C. sinensis cv. Fuding-dabaicha in the field. Mycelial plugs (6-mm diam.) and a conidial suspension (106 conidial/mL) were applied on punctured leaves using a sterile needle and non-punctured leaves. Inoculation with only a PDA plug or sterile water served as controls. Brown spots appeared on the wounded sites of tea leaves at 2 days post-inoculation. No symptoms were observed on the non-wounded leaves or wounded leaves inoculated with PDA plugs lacking mycelia. The re-isolated pathogen from diseased plants was identical to the purified strain ACCC39731 used for inoculation, with re-isolation frequency being 85.0%. To our knowledge, this is the first report of E. nigrum causing leaf spot on tea plants in China, and our findings will be useful for its management and further research.