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.

Optimization of the Extraction Process and Application of Bacterial Extracts in the Control of Brown Spot and Leaf Blast in Rice Culture

The objective of this work was to optimize the extraction process and application of bacterial extracts of Bacillus sp. and Serratia sp. in leaf blast control (Magnapothe oryzae) and brown spot (Bipolaris oryzae) in rice culture. The work was divided into three stages: 1) Bacterial obtaining extracts through liquid-liquid extraction 2) Antagonistic capacity of bacterial extracts to M. oryaze and B. oryae 3) Suppression of brown spot (A1) and leaf blast (A2) in greenhouse. The bacterial isolates in present study were identified as Bacillus sp. (BRM32110) and Serratia marcescens (BRM32113). The crude extract of both isolates at different extraction times 6, 16, 24, 48 and 72 hours reduced the growth of colonies of M. oryzae and B. oryzae by up to 92% and 28%, respectively. The extracts that showed highest inhibition of colony growth were those obtained after 6 and 16h of incubation and were selected for subsequent assays. These, for both isolates were able to reduce conidia germination by up to 91% and apressorium formation of M. oryzae by up to 93%. In green house, A1 the treatment that stood out was the extract of Bacillus sp. (16h) with 6.7% of leaf area affected and in A2 the treatment S. marcescens extract (16h) stood out with only 7.6% of leaf area affected with brusone when compared to control. The use of extracts of Bacillus sp. and Serratia marcescens was efficient in reducing the severity of brown spot and leaf blast in rice crop.

Biochemical Profiling of Resistant Phenotypes Against Bipolaris oryzae Causing Brown Spot Disease in Rice

Brown leaf spot disease of rice is a dominant lethal disease, caused by the fungus Bipolaris oryzae. The pathogen is an obligate parasite and causes qualitative and quantitative damage to rice crop. The objective of the present study was to investigate what extent the defense related biochemical compounds reflect the distinct categories of resistance phenotypes in rice against brown spot disease. This was done by determining the concentration of Catalase (CAT), Phenylalanine ammonia-lyase (PAL), Polyphenol oxidase (PPO), Peroxidase (POD), and β-1,3-Glucanase enzymes in resistant, moderately resistant and susceptible rice genotypes. The disease resistant phenotypes in rice line (PARC-7) reflect the higher accumulation of CAT, PAL, PPO, POD, and β-1,3-Glucanase. The pattern of enzyme accumulation was similar in all resistant genotypes. The rice genotypes with moderately resistant phenotypes showed significant difference with respect to the concentration of biochemical defense-related compounds. The difference in accumulation of defense related enzymes reflected the level of disease severity (% leaf area covered) on resistant and moderately resistant genotypes. The susceptible rice genotypes showed the minimum concentration of these enzymes, with the lowest concentrations found in the rice variety Bas-2000 (80% Disease Index). The differential defense response in resistant and susceptible genotypes suggests that these enzymes can be used as biochemical markers for early detection of disease resistant genotypes. The study of enzyme accumulation at different time points and at different levels of disease severity helps to understand the resistance mechanisms against brown spot disease in rice.

Reaction of some rice (Oryza sativa L.) varieties to brown spot disease caused by Bipolaris oryzae (Breda de Haan) Shoemaker

In Africa, rice is produced and is a source of food energy in most developing countries. But its cultivation faces to brown spot disease, caused by Bipolaris oryzae (Breda de Haan) Shoemaker, which is a serious seed-borne and seed transmitted disease of rice worldwide.The aim of this study was to screen rice varieties for resistance to brown spot disease in field conditions inYaoundé. A completely randomized Fisher block design with three repetitions was used during 2 growing seasons (2015 and 2016). Growth, yields and disease (incidence and severity) parameters were evaluated on four rice varieties (Nerica 3 and 8, White rice and Kamkou). Results show that height of the Nerica 8 variety (86.03cm) and the number of tillers of Kamkou variety (21.66) were significantly highest compared to others varieties (P<0.05) at 59 Days After Sowing (DAS). There was no significant difference on disease incidence for the different rice varieties at the end of rice plant cycle. However disease severity was lower on Nerica 3(2.71%) and Nerica8 (2.05%) varieties and higher on White variety (4.57%). Hence, Nerica 3 and 8 varieties were more tolerant to brown spot disease than others varieties. Overall, Nerica 3 (3.68 t ha-1) and Kamkou (3.51 t ha-1) varieties resulted in higher yields than white rice 28.93 t ha-1.

ANTIFUNGAL ACTIVITY OF Heteranthera reniformis EXTRACTS AGAINST Bipolaris oryzae1

ABSTRACT Plants synthesize specialized metabolites to defend themselves against pathogens. These active compounds, when isolated and identified, can be used as template for fungicide development. Based on field observations, Heteranthera reniformis (kidney leaf mudplantain) could potentially synthesize compounds with antifungal activity. The goal of this study was to evaluate the fungicidal activity of H. reniformis leaf extracts on Bipolaris oryzae development. The activities of hexane, ethyl acetate, and methanol extracts of H. reniformis leaves were tested on mycelial growth, sporulation, and colony morphology. Due to the highest inhibition of B. oryzae sporulation, methanol extract was chosen for concentration tests. The effect of methanol extract on B. oryzae spore length and germination was also evaluated. Methanolic extract was the most active in inhibiting B. oryzae sporulation. The morphology of the colonies is altered when the fungus grows in medium containing H. reniformis leaf extracts. Higher concentration of methanol extract stimulates mycelial growth and suppresses B. oryzae sporulation. There are reductions in length and number of germinated B. oryzae spores caused by methanol extract of H. reniformis leaves. Methanolic extract has compounds with antifungal activity and should be subjected to bioassay-guided isolation for purification and identification of these active compounds.

Breeding and Characterization of The World’s First Practical Rice Variety With Resistance to Brown Spot (Bipolaris Oryzae)

Background: Brown spot (BS) caused by Bipolaris oryzae is a serious disease of rice and decreases grain yield. Breeding for BS resistance is an economical solution but has not hitherto been achieved.Results: To develop a practical BS-resistant variety, we introduced a chromosomal segment including a quantitative trait locus (QTL) for BS resistance, qBSfR11, derived from the BS-resistant local resource ‘Tadukan’, into the genetic background of the high-yielding but susceptible ‘Mienoyume’. Resistance is controlled by a single recessive gene in a 1.3-Mbp region. We named this gene bsr1 (brown spot resistance 1). The near-isogenic line bsr1-NIL had a greater yield with larger grain width than Mienoyume but similar other agronomic traits in fields where BS was mild; it had a significantly lower BS disease score and a 28.8% higher yield in fields where BS was more severe, and it showed resistance to multiple isolates of BS fungus. It showed stable resistance to BS and had excellent agricultural traits in the presence of BS.Conclusions: We developed the bsr1-NIL with resistance to BS and applied it for variety registration to Ministry of Agriculture, Forestry and Fisheries in Japan as ‘Mienoyume BSL’. It is the world’s first practical breeding variety with resistance to BS.

Longevity of Bipolaris oryzae on HEERA-2 Hybrid Rice Seed

Seeds of hybrid rice variety Heera-2 was collected from Mymensingh district in Bangladesh. Longevity of Bipolaris oryzae was studied in 2012 to 2016 at Seed Pathology Centre, Bangladesh Agricultural University, Seed processing &preservation center and Seed Pathology Laboratory of Supreme Seed Company Limited. The data collected from the experiments were analyzed for test of significance and compared the treatment means by using DMRT at 5% level of probability following the Statistical tool for Agricultural Research (STAR) 2.1 program.The longevity of B. oryzae was observed till five years by blotter incubation methods, seedling symptom test and growing on test. Both untreated and seeds treated with Vitaflo 200FF (carboxin and thiram) were kept in jute bag and polybag stored for five years under both normal and dehumidified condition. The inocula of B. oryzae on Hybrid Heera 2 seed remained viable up to five years. When the seed samples were stored under dehumidified condition, the highest incidence of B. oryzae was recorded in case of jute bag after five years. After treated with Vitaflo 200FF, there was no any infection of B. oryzae under both storage condition Environ. Sci. & Natural Resources, 12(1&2): 209-213, 2019

First Report of Bipolaris oryzae Causing Leaf Spot on Cultivated Wild Rice (Oryza rufipogon) in China

Wild rice (Oryza rufipogon) has been widely studied and cultivated in China in recent years due to its antioxidant activities and health-promoting effects. In December 2018, leaf spot disease on wild rice (O. rufipogon cv. Haihong-12) was observed in Zhanjiang (20.93 N, 109.79 E), China. The early symptom was small purple-brown lesions on the leaves. Then, the once-localized lesions coalesced into a larger lesion with a tan to brown necrotic center surrounded by a chlorotic halo. The diseased leaves eventually died. Disease incidence was higher than 30%. Twenty diseased leaves were collected from the fields. The margin of diseased tissues was cut into 2 × 2 mm2 pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s, and then rinsed three times with sterile water before isolation. The tissues were plated on potato dextrose agar (PDA) medium and incubated at 28 °C in the dark for 4 days. Pure cultures were produced by transferring hyphal tips to new PDA plates. Fifteen isolates were obtained. Two isolates (OrL-1 and OrL-2) were subjected to further morphological and molecular studies. The colonies of OrL-1 and OrL-1 on PDA were initially light gray, but it became dark gray with age. Conidiophores were single, straight to flexuous, multiseptate, and brown. Conidia were oblong, slightly curved, and light brown with four to nine septa, and measured 35.2–120.3 µm × 10.3–22.5 µm (n = 30). The morphological characteristics of OrL-1 and OrL-2 were consistent with the description on Bipolaris oryzae (Breda de Haan) Shoemaker (Manamgoda et al. 2014). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor (EF-1α) were amplified using primers ITS1/ITS4, GDF1gpp1/GDR1 gdp2 (Berbee et al. 1999), and EF-1α-F/EF-1α-R EF-1/EF-2 (O’Donnell 2000), respectively. Amplicons of OrL-1 and OrL-2 were sequenced and submitted to GenBank (accession nos. MN880261 and MN880262, MT027091 and MT027092, and MT027093 and MT027094). The sequences of the two isolates were 99.83%–100% identical to that of B. oryzae (accession nos. MF490854，MF490831，MF490810) in accordance with BLAST analysis. A phylogenetic tree was generated on the basis of concatenated data from the sequences of ITS, GAPDH, and EF-1α via Maximum Likelihood method, which clustered OrL-1 and OrL-2 with B. oryzae. The two isolates were determined as B. oryzae by combining morphological and molecular characteristics. Pathogenicity test was performed on OrL-1 in a greenhouse at 24 °C to 30 °C with 80% relative humidity. Rice (cv. Haihong-12) with 3 leaves was grown in 10 pots, with approximately 50 plants per pot. Five pots were inoculated by spraying a spore suspension (105 spores/mL) onto leaves until runoff occurred, and five pots were sprayed with sterile water and used as controls. The test was conducted three times. Disease symptoms were observed on leaves after 10 days, but the controls remained healthy. The morphological characteristics and ITS sequences of the fungal isolates re-isolated from the diseased leaves were identical to those of B. oryzae. B. oryzae has been confirmed to cause leaf spot on Oryza sativa (Barnwal et al. 2013), but as an endophyte has been reported in O. rufipogon (Wang et al. 2015).. Thus, this study is the first report of B. oryzae causing leaf spot in O. rufipogon in China. This disease has become a risk for cultivated wild rice with the expansion of cultivation areas. Thus, vigilance is required.