Alternaria Brassicae Research Articles

Development of multiplex PCR assay for detection of Alternaria brassicae, A. brassicicola and Xanthomonas campestris pv. campestris in crucifers.

Among biotic stresses, Alternaria leaf spots caused by Alternaria brassicae and A. brassicicola and black rot caused by Xanthomonas campestris pv. campestris are major limiting factors in brassica cultivation across the world. Because of seed-borne nature of these pathogens primarily, disease-free conservation as well as exchange of brassica seeds at domestic as well as international level are major challenges. To facilitate disease-free conservation and transboundary movement of brassica germplasm, a highly specific and sensitive method was developed for simultaneous detection of these pathogens. A set of primers namely, AbeABC1F and AbeABC1R based on ABC transporter (Atr1) gene for A. brassicae, Aba28sF and Aba28sR based on SSR marker was developed for A. brassicicola as well as rpf gene-based primers namely, rpfH_F and rpfH_R for X. campestris pv. campestris were used for multiplex PCR. The specific bands of 586, 201 and 304bp were obtained in multiplex PCR assay for A. brassicae, A. brassicicola and X. campestris pv. campestris, respectively. Therefore, the developed multiplex PCR protocol could be utilized for a reliable diagnosis of these pathogens to facilitate safe conservation, exchange of seeds to the researchers and also by seed certification agencies for ensuring quality seed availability to farmers.

Epidemiological models based on meteorological variables to forewarn Alternaria blight of rapeseed-mustard

Alternaria blight [Alternaria brassicae (Berk.) Sacc.] is one of the most widespread and harmful maladies of rapeseed-mustard, causing yield loss up to 47 per cent. Meteorological parameters especially temperature, relative humidity and bright sunshine hours play major role in the development of Alternaria blight disease. Infection by the pathogen is highly influenced by meteorological conditions. A well-tested model based on meteorological variables is an efficient tool for forewarning this disease. Epidemiology of Alternaria blight of brassicas was investigated based on long term data during 2003-2018 crop seasons on the disease severity and meteorological variables, which was validated with data for two subsequent years. During this study, meteorological variable-based regression model of forewarning was developed for maximum severity (%) of Alternaria blight on leaves and pods for three locations viz., New Delhi, Hisar (Haryana) and Mohanpur (West Bengal)] in India. Validation of the forewarning models for maximum severity (%) of Alternaria blight proved the efficiency of the targeted forecasts.

First Report of Leaf Spot Disease Caused by Alternaria brassicae on Orychophragmus violaceus in China.

Orychophragmus violaceus, belonging to the Brassicaceae family, is widely grown in many provinces of China as an ornamental plant and also as a green manure crop. In December 2019, field investigations showed that a leaf spot disease occurred on O.violaceus with 50% to 80% incidence in Huize City, Yunnan Province of China. Infected leaves showed symptoms of small black point spots in the early stage of onset. The lesions are distributed throughout the leaves and finaly expand to 10-15 mm in diameter after 10-15 days of onset. At this time, the lesions are gray to black, and some have round patterns, and gray-white mildew layers can be seen on the front and back of the lesions in a humid environment. The leaves with typical lesion symptoms were sampled and photographed, and then subjected to isolate and characterize the pathogen. Six pure cultures (HEYA2; HEYA4; HEYC6; HEYD7; HEYD8; HEYD10) were obtained by single-hyphae isolation. On PCA medium, colony can reach 27 mm after 7d, at 25°C in darkness. Aerial hypha is cottony with white to pale gray color, while the colony reverse is fawn to dark. on V8 medium, conidiophore solitary or clustered, erect or knee-curved, occasionally branched, pare brown, separated, 82-130 × 5-9 µm. Conidia are solitary, straight or slightly curved, inverted rod-shaped, pare brown to brown, with 6-10 transverse septa, 0-5 oblique and longitudinal septa, columnar beak, conidial bodies (47.7-)69.3-103.8(-119.6)(11.2-)16.6-23.6(-27.8) µm. Beak septum, pare brown, (29.2-)34.4-72.4(-101.3)(4.2-)6.6-9.5(-11.3) µm. Morphologically these isolates resembled species belonging to genus Alternaria (Simmons, 2007). Genomic DNA of each culture was quickly extracted from mycelia using QS method (Chi et al. 2009). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2) and translation elongation factor -1α (TEF -1α) genes were amplified according described procedures (White et al. 1990; Berbee et al. 1999; Liu et al. 1999；Sung et al. 2007; Carbone & Kohn 1999). The sequences obtained in this study were deposited in GenBank with accession numbers: MW867245, MW867246, MW867247, MW867248, MW867249, MW867250, MW882913, MW882914, MW882915, MW882916, MW882917, MW882918, MW882919, MW882920, MW882921, MW882922, MW882923, MW882924, MW882925, MW882926, MW882927, MW882928, MW882929, MW882930. Phylogenetic analysis was conducted with combined sequences of the four loci, using the maximum likelihood method and the maximum parsimony method. In the phylogenetic tree, the six isolates and Alternaria brassicae (CBS 116528) clustered together with high bootstrap support values (MLBS=100; MPBS= 100). Based on both morphological characters and phylogenetic results, the isolates were identified as Alternaria brassicae. Pathogenicity test of isolate HEYA2 was carried out on the detached leaves in a dark thermostat incubator at 25°C. Five pots per leaf were inoculated with mycelia plugs (5 mm in diameter), another five pots were inoculated with pure agar plugs and used as the negative control. In addition, conidia suspension (105 conidia/ml) of isolate HEYD8 were sprayed on 3-month-old healthy plants grown in a greenhouse at 22 °C-28 °C. The plants sprayed with sterilized water were used as negative controls. The test was conducted three times. After 5-7 days, the leaves inoculated with other the conidia suspension or the mycelium plugs showed brown necrotic lesions that are similar to the symptoms observed in the field, but the controls remained healthy. The pathogen was reisolated and confirmed to be A. brassicae, completing Koch's postulates. To our knowledge, this is the first report of leaf spot disease caused by A. brassicae on Orychophragmus violaceus in China.

Anti-fungal evaluation of synthesized 1,3,5-triazine derivatives against phytopathogenic fungi

Seven mono-, di- and tri-substituted derivatives of s-triazine were synthesized by condensation reaction of s-triazine with different aromatic amines at different temperature conditions. The synthesized compounds were characterized by various spectral techniques, i.e. 1HNMR, 1HNMR-D2O, 13CNMR, elemental analysis and mass spectrometry and screened for their antifungal activity against Helminthosporium oryzae, Rhizoctonia solani and Alternaria brassicae. Among all the synthesized triazine derivatives; compounds (5c), (7b) and (5a) were found to be the most effective with ED50 values 4, 7 and 9 pg/ml against H. oryzae, R. solani and A. brassicae, respectively.

Composition of the Microbiomes from Spinach Seeds Infested or Noninfested with Peronospora effusa or Verticillium dahliae

The worldwide distribution of plant seeds can disseminate beneficial and plant-pathogenic microorganisms. This phenomenon is of particular concern where seed production is geographically isolated from crop production, as is the case with spinach in the United States. We aimed to characterize the structure and function of spinach seed microbiomes in commercial spinach seed lots originating from Europe and New Zealand. The seed lots we analyzed were infested with Peronospora effusa and Verticillium dahliae, only infested with V. dahliae, or not infested with either of these pathogens. The microbial taxonomic composition and gene function (assessed by gene ontology [GO] terms) of spinach seeds were highly influenced by geographic origin and the status of pathogen infestation. Through taxonomic profiling, we found that potentially plant-beneficial bacterial genera such as Pseudomonas and Pantoea were the most abundant taxa both in infested and noninfested seeds, and Stenotrophomonas was observed in seed lots infested with P. effusa and V. dahliae. Many potential plant pathogens that are not known to be associated with spinach seed were also discovered by metagenomic analysis, including Sclerotinia sclerotiorum, Botrytis cinerea, Bipolaris sorokiniana, Fusarium pseudograminearum, Alternaria brassicae, A. solani, Parastagonospora nodorum, and Pyrenophora teres f. teres. Our analysis of the function of prokaryotic genes in de novo assembled metagenomes revealed distinct GO terms associated with the geographic origin of seed lots. This work provides an important first step toward identifying spinach seedborne microorganisms that could be utilized to improve plant health and plant pathogens that could be inadvertently carried to new locations.

ANTIFUNGAL ACTIVITY OF INVASIVE ALIEN PLANT SPECIES (IAPS) – IMPLICATION FOR MANAGEMENT

The antifungal activity of aqueous and methanolic extract of six invasive alien plants species (IAPs) Ageratina adenophora, Ageratum houstonianum, Parthenium hysterophorus, Xanthium strumarium, Ipomoea carnea and Mikania micrantha was investigated against Alternaria brassicae, Botrytis cinerea, Fusarium oxysporum, Phytophthora capsici and Sclerotium rolfsii at different concentrations (5, 10, 15 , 20 and 25%). Methanolic extract of plant exhibited strong inhibitory effect than aqueous extract. At 25% maximum antifungal potential was observed with the Methanolic extracts of Ageratum houstonianum and Xanthium strumarium against F. oxysporum (100%), P. capsici (100%), S. rolfsii (100%), followed by A . adenophora, which recorded excellent inhibitory activity against F. oxysporum(100%) P. capsici (100 %) and S. rolfsii (73%). Likewise methanolic extract of I. carnea at 25%, recorded maximum inhibitory activity against F. oxysporum (100%) which was followed by A. brassicae (64.0%), extract of Parthenium hysterophorus had shown excellent inhibitory activity against Fusarium oxysporum (100%) and S. rolfsii (100%). Extract of Mikania micrantha exhibited inhibitory effect on B. cinerea (61.50%), followed by F. oxysporum (61.3%). Test revealed the presence of tannins, cardiac. glycosides, alkaloids, and flavanoids in all the tested extracts. This study demonstrated that all studied plant species could be used as a potential fungicides in plant disease management. Utilization of IAPs as antifungal agent, could be helpful for its management

Effect of Selected Essential Oils and Botanicals on Alternaria Blight (Alternaria brassicae (Berk) Sacc) of Mustard (Brassica juncea (Linn) Czern and Coss

Indian mustard (Brassica juncea (L.) Czern.coss) is also known as sarson, rai or raya, toria or Lahi.it is a herbaceous annual plant. Mustard is the second important oilseed crop in the world after sunflower, soybean and palm oil. Alternaria blight disease caused by Alternaria brassicae (Berk.) Sacc. It has been reported from all the continents of the world. Average yield losses occur due to various pest and diseases among which Alternaria blight is an important disease. Field experiment was conducted at the research plot of the Department of Plant Pathology, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj, Uttar Pradesh during the rabi season of 2020-2021 to test, Effect of selected essential oils and botanicals against Alternaria blight (Alternaria brassicae (Berk.) Sacc) of mustard (Brassica juncea (Linn.) Czern and Coss), by foliar spray of certain essential oil, plant extracts and fungicide. The treatments were Neem oil @2%, Eucalyptus oil @2%, Pongamia oil @2%, Lawsonia inermis extract @15%, Chenopodium album extract @15% Mancozeb (treated check) @ 0.2% and control (untreated check). The percent disease intensity on leaves at 45, 60 and 75 DAS, number of siliquae per plant, number of seeds per siliquae, length of siliquae (cm), test weight of seed (1000 number), biological yield (gm) and seed yield were recorded. Among the treatments, maximum number of number of siliquaes (243.13), maximum number of seeds per siliquae (12.27), length of siliquae (5.16 cm), minimum disease intensity (%) (29.01 %), maximum test weight (3.57 gm), maximum yield (9.43 qt) and biological yield (24.70 gm) were recorded in the treatment T2 Eucalyptus oil @2% followed by T1 Neem oil @2%, T4 Lawsonia inermis extract @15%, T3 Pongamia oil @2%, T5 Chenopodium extract @15%, when compared to treated check T6 Mancozeb @ 0.2% and untreated check T0. Higher gross return value (Rs. 66295), net return value (Rs. 29295), and B: C ratio (1.7:1) was found in the treatment T2 – Eucalyptus oil @2% as compared to T5–mancozeb and T0–control.

Antimicrobial and Antioxidant properties of medicinal mushroom Ganoderma P. Karst

Ganoderma species have been known all over the world as highly medicinal mushrooms. Antimicrobial activity of it is an attractive approach which raises the global interests from the scientific community. In this study, the antimicrobial assay of ethanol and methanol extracts of Ganoderma were prepared by using the dried mycelial powder obtained from five different liquid media; was performed against seven plant pathogenic fungi viz., Alternaria macrospora, Aspergilus niger, A. flavus, Colletotrichum falcatum, Fusarium oxysporum, Pestalotiopsis mangiferae and Penicillium sp. and two plant pathogenic bacteria Xanthomonas oryzae and Ralstonia solanacearum. The extracts of mycelia obtained from Yeast Wine Media exhibited the highest inhibition percentage as compared to rest. At concentration 1000ppm, there was complete inhibition of mycelial growth for Alternaria macrospora, Aspergilus niger and A. flavus while for Fusarium oxysporum and Penicillum sp. complete inhibition was observed at 500ppm but for Colletotrichum falcatum and Pestalotiopsis mangiferae, more than 80% mycelial inhibition at concentration 1000ppm in both ethanol and methanol extracts. In the case of Xanthomonas oryzae and Ralstonia solanacearum, at concentration 1000ppm, methanol extract showed the highest inhibition zone (3.50mm, 3.75mm). Ganoderma exhibited antagonistic effect against plant pathogenic fungi could add to the interest of developing Ganoderma as a successful bioagent in the near future. Antioxidant activities were evaluated by using DPPH radical scavenging assay and Metal chelating activity on ferrous ions. The DPPH radical scavenging effect was detected in methanol extract (Inhibition% = 27.312%) was higher than that of the ethanol extract (Inhibition% = 24.79%) and also Ferrous ion chelating ability of methanol extract (Inhibition% =22.27%) was higher than the ethanol extract (Inhibition% =12.55%). It is clearly indicated that both methanol and ethanol extract of the Ganoderma show antioxidant properties and Ganoderma extracts act as an effective antioxidant agents.

Novel biosynthesis of Ag-nanocomplex for controlling Verticillium wilt disease of olive tree

ABSTRACT The nano-metal complexes are found to be more impregnable than their parent drugs, therefore this study was aimed to synthesize Ag-nanocomplex of paracetamol (Para) by novel biosynthesis method. Paracetamol Ag-nanocomplex with the formula [Ag(Para)2(NO3)2] has been characterized and synthesized on the basis of elemental analysis, thermal (TGA), conductivity, 1H, 13C NMR and electronic spectral studies. Verticillium wilt, caused by Verticillium dahliae is considered one of the most serious diseases in olives. The use of Ag-nanocomplex as antifungal with different concentration (25, 50, 100 and 150 mg/ml) led to significant reductions of forming colonies of Verticillium wilt pathogenic fungi, with efficiencies reaching to 79.4, 82.9 and 81.8%, respectively at (100 mg/ml) against “V. dahliae, Fusarium acuminatum and Alternaria brassicae” LD50 was also evaluated, while no changes in physical activity were detected, so this research opens outlooks for more study of fungal biogenic nanoparticles, indicating their use for controlling an agricultural pests

New and potent production platform of the acetylcholinesterase inhibitor huperzine A by gamma-irradiated Alternaria brassicae under solid-state fermentation.

Huperzine-A (HupA) is an emerging, powerful, and promising natural acetylcholinesterase inhibitor. Despite that, the achieved yields of HupA from microbial sources are still far from the industrial applications. Accordingly, this paper was conducted to valorize solid-state fermentation (SSF) as an efficient production platform of HupA. Four agro-industrial wastes, namely rice bran, potato peel, sugarcane bagasse, and wheat bran, were tested and screened as cultural substrates for the production of HupA by the endophytic Alternaria brassica under SSF. Maximum HupA production was attained on using rice bran moistened by Czapex's dox mineral broth. In the effort to increase the HupA titer, supplementation of the best moistening agent by different carbon and nitrogen sources was successfully investigated. Additionally, factors affecting HupA production under SSF including substrate concentration, moistening level, and inoculum concentration were optimized using response surface methodology. A Box-Behnken design was applied for generating a predictive model of the interactions between these factors. Under the optimum conditions of 15g rice bran, inoculum concentration of 5 × 106 spores mL-1, and 60% moisture level, HupA concentration was intensified to 518.93μgg-1. Besides, HupA production by the fungal strain was further enhanced using gamma-irradiation mutagenesis. The final HupA production was significantly intensified following exposure to 0.5 KGy gamma radiation to 1327μgg-1, which represents a 12.85-fold increase. This is the first report on the successful production of the natural fungal metabolite HupA under SSF. Moreover, the achieved yield in this study using agro-industrial wastes may contribute to reducing the cost of HupA manufacture.Key points• Different agro-industrial by-products were tried as cultural substrates for the production of the acetylcholinesterase inhibitor HupA under SSF for the first time.• Factors affecting HupA production under SSF were optimized using response surface methodology.• The final HupA production was intensified following exposure to gamma radiation recording 1327μgg-1, which represents a 12.85-fold increase.

Synergistic/antagonistic interactions between Neopseudocercosporella, Alternaria, Leptosphaeria, and Hyaloperonospora determine aggregate foliar disease severity in rapeseed

AbstractWhite leaf spot (Neopseudocercosporella capsellae), Alternaria leaf spot (Alternaria brassicae), blackleg (Leptosphaeria maculans), and downy mildew (Hyaloperonospora brassicae) commonly co‐occur on rapeseed (Brassica napus). In controlled environment studies, the synergistic/antagonistic interactions between these four pathogens were determined using two cultivars (Surpass 400, Thunder TT) with contrasting susceptibilities/resistances towards these pathogens. A spectrum of significant (p < 0.001) contrasting synergistic/antagonistic interactions between the four pathogens was observed and specific pathogen–pathogen interaction outcomes were determined by sequence of inoculation of different pathogens on each cultivar. On Thunder TT, Alternaria leaf spot was more severe (p < 0.001) when A. brassicae was applied prior to N. capsellae, but was reduced (p < 0.001) when A. brassicae was applied following N. capsellae. On Surpass 400, Alternaria leaf spot was enhanced (p < 0.001) when A. brassicae was applied following N. capsellae. For interactions involving H. brassicae on Thunder TT, downy mildew was reduced (p < 0.001) when co‐inoculated with N. capsellae, A. brassicae, or L. maculans, irrespective of co‐inoculating pathogen application sequence. This study highlights how disease severity of one or more co‐occurring pathogens is enhanced/decreased in the presence of other co‐occurring pathogens and emphasizes the importance of both pathogen inoculation sequence and relative host plant resistances/susceptibilities to different pathogens in together determining the total disease burden from these co‐occurring rapeseed diseases. Clearly, there is a need for rapeseed germplasm selection and breeding programmes to seriously consider the implications of co‐occurring pathogen complexes rather than solely relying upon single‐pathogen screening tests.

EFFECT OF COW URINE TREATMENT ON PLANT GROWTH AND ANTI-MICROBIAL ACTIVITY ON GOSSYPIUM HIRSUTUM L.

In Hindu culture, the cow is treated as a divine animal. Cow's urine, cow dung and Milk are utilized for different purposes. Cow's urine has been utilized as an enhancer of plant growth and an antifungal agent for the current study. The plant selected for this study Gossypium hirsutum L.(cotton).The plant was grown utilizing various concentrations of cow urine, such as 0% (control), 1:10, 1:5, 1:1 for 90 days. Various parameters for example leaf area, shoot length, plant height, root length, as well as biomass of the plant have been monitored. The antifungal activity has been studied by utilizing different concentrations of cow urine against (Alternaria macrospora) using agar diffusion method. Among all the concentrations, the 1:1 concentration showed maximum plant growth compared to other two concentrations. On the other hand, 1:5, 1:10 concentrations showed more antifungal activity. The conclusion of this study found that cow urine can be utilized as a growth enhancer of plant and has antifungal properties.A

Application timing of herbicides, glyphosate and atrazine, sway respective epidemics of foliar pathogens in herbicide‐tolerant rapeseed

AbstractRapeseed (Brassica napus) production in Australia relies heavily on triazine‐or glyphosate‐tolerant cultivars. For 14 triazine‐tolerant cultivars, disease development of Neopseudocercosporella capsellae (white leaf spot), Alternaria brassicae and A. japonica (Alternaria leaf spot), and Hyaloperonospora brassicae (downy mildew) were all dependent upon herbicide application timing (p < 0.001), with significant differences between cultivars (p < 0.001) and a significant interaction (p < 0.001) between herbicide application timing and cultivars. Atrazine applied preinfection by N. capsellae, A. brassicae, or A. japonica enhanced disease incidence, severity, and leaf collapse, while atrazine application postinfection for these same pathogens reduced all three disease parameters. However, for H. brassicae, application of atrazine after, and especially prior to, infection resulted in lower disease incidence, severity, and leaf collapse. Application of glyphosate on five glyphosate‐tolerant cultivars for N. capsellae resulted in significant differences (p < 0.05) between glyphosate application treatments, and between host cultivars in terms of incidence and consequent leaf collapse. For A. brassicae, A. japonica, and H. brassicae, glyphosate resulted in significant differences (p < 0.001) across application timings between cultivars, and a significant interaction (p < 0.001) between herbicide application timings and cultivars. Glyphosate applied on glyphosate‐resistant rapeseed after, and especially prior to, attack by H. brassicae, reduced downy mildew. These are the first studies to highlight how the timing of application of triazine or glyphosate in relation to pathogen infection is critical to the susceptibility of rapeseed to white leaf spot, Alternaria leaf spot, and downy mildew. This new understanding offers fresh possibilities for improved management of these diseases in herbicide‐tolerant rapeseed crops.

Introgression and QTL mapping conferring resistance for Alternaria brassicae in the backcross progeny of Sinapis alba + Brassica juncea somatic hybrids.

A total of three QTLs, responsible for A. brassicae resistance were introgressed into S. alba - B. juncea introgression lines from S. alba and mapped through donor genome-specific SSR markers. Alternaria brassicae is a key pathogen of the Brassicaceae family causing severe blight disease to oilseed crops that leads to heavy yield losses due to lack of resistance source within cultivated Brassicas. However, the host resistance present in the Sinapis alba, an allied member of the Brassicaceae family is still unexplored precisely due to the unavailability of segregating population for Alternaria blight resistance and scarcity of donor genome-specific genetic markers. The present study was undertaken to identify quantitative trait loci governing resistance to Alternaria blight which was introgressed from S. alba to the backcross population of stable S. alba + B. juncea somatic hybrids (2n = 60; AABBSS). The second backcross population showed significant phenotypic variations for Alternaria blight ranging from immune to highly susceptible phenotype, thus suggesting quantitative nature of resistance for the disease. A subset of 154 BC2F3-4 lines was used for disease screening and genotyping with 234 S. alba genome-specific microsatellite markers. As a result of the study, twelve linkage groups were developed corresponding to 12 chromosomes of S. alba (n = 12) covering a length of 1694.02cM. The chromosomes 5 and 11 harbored a total of 1 (Abr-01), and 2 (Abr-02, and Abr-03) QTLs detected by ICIM-ADD mapping method at LOD score values 3.7, 5.12, and 6.74, respectively. The QTLs identified during the study have a range of 5.51-10.87 percent phenotypic variations for disease resistance. To the best of our knowledge, this is the first report of QTLs introgression for A. brassicae resistance in cultivated Brassica from an allied member of Brassicaceae.

Alternaria Black Spot (Alternaria brassicae) Infection Severity on Cruciferous Oilseed Crops

The increase in the cultivation area of cruciferous oilseed crops and the use of short crop rotation has resulted in the enhanced spread of several major pests in Northern latitudes. There is currently limited information about incidence and severity of Alternaria black spot disease (Alternaria brassicae) on the main oilseed crop, spring oilseed rape (Brassica napus), in the Northern Baltics. Thus, spring oilseed rape and five alternative cruciferous oilseed crops were selected and their resistance to black spot disease was evaluated in field conditions during two growing seasons. We hypothesized that spring oilseed rape is more susceptible to Alternaria black spot disease than other alternative cruciferous oilseed crops. Both growing seasons were warmer and drier compared to the long-term average, and were thus suitable for A. brassicae development and assessments. In both years, incidence of Alternaria black spot infection was recorded on all cruciferous species, yet the disease development differed considerably among the crops. During both growing seasons, black mustard (B. nigra) plants were the most infected. Based on our observations during warm growing seasons we conclude that alternative oilseed crops such as Sinapis alba, Eruca sativa and Raphanus sativus are more resistant to the Alternaria black spot infection than the traditional oilseed crops and thus, possess a great potential to grow with limited chemical disease control in Northern Baltic conditions.

Yield loss assessment due to Alternaria blight disease in rapeseed and mustard: Yield loss due to alternaria Blight in rapeseed and mustard

Alternaria blight disease in rapeseed-mustard caused by Alternaria brassicae and A. brassicicola is the important disease of rapeseed-mustard crop in India. The experiment was conducted to assess the yield losses due to Alternaria blight in different released cultivars of rapeseed-mustard during Rabi 2016-17 and 2017-18 at Student’s Instructional Farm of the A. N. D. University of Agriculture and Technology, Kumarganj, Ayodhya by planting 15 varieties of the rapeseed-mustard namely RH-749, Pusa gold, KBS-3, GSL-5, GSL-2, Pusa mustard-27, Jhumka, Anuradha, PT-30, T-27, PM-25, Pusa aditya, PT-303, Sheetal, TMLC-2 in six replications (three protected and three unprotected under natural conditions). Seed of each variety were treated with Apron @ 6g/kg and three spray of Trifloxystrobin 25% + Tebuconazol 50% (Nativo) @ 0.08% in protected replication. The minimum disease severity on leaves and pods were recorded in protected and unprotected conditions in the variety GSL-5 (2.65% and 16.26%), (1.75% and 6.32%) followed by GSL-2 (3.20% and 19.72%), (1.89% and 6.75%) Pusa aditya (6.91% and 19.89%), (2.18% and 7.20%) and Sheetal (7.99% and 23.13%), (3.21% and 7.43%), respectively. On the basis of mean values the minimum infection rate on pods was recorded GSL-2 (0.083 and 0.042) followed by Pusa aditya (0.089 and 0.045), respectively. The minimum AUDPC on leaf under protected and unprotected was recorded on cultivar GSL-5 (76.40 and 496.20) followed by GSL-2 (88.80 and 596.85), Pusa aditya (205.35 and 588.25), Sheetal (241.70 and 717.30) and RH-749 (244.95 and 950.85), respectively. Whereas the minimum AUDPC on pod was recorded with variety GSL-5 (32.10) and Pusa aditya (200.25) followed by [GSL-2 (34.65) and Sheetal (206.65)], [Pusa aditya (39.35) and RH-749 (566.41)] and [Sheetal (60.95) and Pusa mustard-27 (741.75)], respectively. The maximum yield avoidable loss (%) was recorded T-27 (46.67%) followed by Jhumka (44.53%, Anuradha (40.00%), KBS-3 (38.77%) and Pusa gold (34.13%), respectively. Minimum avoidable loss (%) was recorded on cultivar GSL-5 (11.09%) followed by Anuradha (14.36%), GSL-2 (17.73%), RH-749 (17.90%) and Pusa aditya, respectively. In present investigation out of 15 genotypes examined for blight resistance, GSL-5, Pusa Aditya, Sheetal and GSL-2 exhibited good level of partial resistance i.e. moderately resistance. Other 11 varieties RH-749, Pusa gold, Pusa mustard-27, Jhumka, Anuradha, PT-30, PT-303, T-27, KBS-3, TMLC-2 and PM-25 were susceptible based on the mean basis severity (PDI), reduced apparent infection rate and lesser value of AUDPC.

Morphological and Molecular Characterization of Alternaria Species Isolated from Different Plants

Alternaria belongs to the family of Pleosporaceae, the order of Pleosporales, the class of Dothideomycetes in the phyllum of Ascomycota. This fungal genus is characterized by its ability to produce a number of enzymes able to injured many types of crops. The symptoms of Alternaria blight on different agricultural crops such as cabbage, cauliflower, coriander, fenugreek, brinjal, onion, faba bean, dahlia, dracaena, hollyhock, carrot, marigold, tomato and wheat were observed. Alternaria produces distinctive "bulls eye" patterned leaf spots in almost all the infected plants. Nineteen Alternaria isolates were obtained from fourteen infected plant samples. The observations were recorded from the seven day old culture for colony characteristics on PDA. Based on morphology 10 isolates of Alternaria alternata, two isolates Alternaria tenuissima and Alternaria longipes and one Alternaria porri, Alternaria brassicae, Alternaria brassicicola and Alternaria sp. were identified. Alternaria species grow rapidly and produced flat, downy to woolly grayish green to black colonies. All the isolates exhibited characteristics dark-coloured multicelled conidia with longitudinal and transverse septa (phaeo-dictyospores) and a beak or tapering apical cells. DNA isolation of all isolates of Alternaria was carried out using CTAB method. Isolated DNA was subjected to amplification with ITS1 and ITS4 primers in a thermal cycler. The amplified products ranged from 580-600bp. These amplified products were sequenced and identify the species of different Alternaria isolates using BLASTn in NCBI online. All the sequences were published in NCBI public domain. The resulting sequences of all isolates were compared to other sequences in the GenBank as 90-100% identical. Genetic variability was conducted by phylogenetic analysis.

Evaluation of Trichoderma-based biopesticides against plant pathogens and agronomic crop response

Biopesticides are the part of bio-inputs driven agriculture, popularly define the success of organic agriculture, especially in northeast India. Even today, many of the disease causing pathogens (Fusarium oxysporum f. sp lycopersicum, Alternaria brassicae, Colletotrichum lindemuthianum, Rhizoctonia solani and Sclerotium rolfsii) are beyond their control with conventional management strategy. In the year 2019-20, an evaluation was carried out in the department of Plant Pathology, Assam Agricultural University, Jorhat, Assam where we collected as many 10 commercial formulations marketed widely as biopesticides, and observed the population of Trichoderma sp. ranging from 1.0×105 to 2.0×108 cfu/g, inclusive of two predominant species T. harzianum and T. viride. As many five formulations, viz. Biogreen, Biofor –PF2, Biolime, Bioveer, Biozium and Biozin-PTB were observed effective in suppressing the growth of these five pathogens upto 91.2-97.1% under challenge inoculation in-vitro studies, which corroborated the response on tomato and rice seeds with regard to germination and vigour index (I and II). Our studies warranted greater effectiveness of these formulations carrying multiple antagonists in addition to strong seed priming effectsof Trichoderma sp.

Molecular modeling, QSAR analysis and antimicrobial properties of Schiff base derivatives of isatin

• New isatin Schiff base derivatives have been synthesized as potent antimicrobial agents. • The compounds were docked at the active site of PDF enzyme (PDB ID: 1G2A ) to predict the possible mode of action of compounds. • Docking studies indicated the stable ligand-protein complexes formed by all compounds. • The TOPKAT protocol in DS 3.0 software showed lower toxicity of the designed isatin Schiff base derivatives. • Antimicrobial study and regression analysis have been done for all compounds. A new series of novel Schiff bases has been designed, synthesized and tested for in vitro antimicrobial activity. QSAR studies followed by antibacterial screening using broth dilution technique showed excellent MIC values against four human pathogens, namely Escherichia coli, Pseudomonas aeruginosa, Bacillus cerus and Staphylococcus aureus. A great synergistic effect was observed when the test compounds were used in combination with chloramphenicol and ciprofloxacin. The MIC of the combination was reduced up to 1/4 th or 1/32 nd of the original value, indicating thereby that the combination was 4 to 32 times more potent than the test compounds. Molecular modeling suggested that all ligands interacted well within the active site of peptide deformylase enzyme (PDF) (PDB ID: 1G2A ) and formed stable complexes. Antifungal screening of all compounds was also done using the food poisoning in vitro method on four plant pathogenic fungi, namely Fusarium oxysporum, Aspergillus flavus, Aspergillus niger and Alternaria brassicae. The compounds 8 and 10 showed good activity against F. oxysporum at 100 µg/mL with inhibitory index 70% and 82.5%, respectively and IC 50 value of 2.75 and 2.29 µg/mL, respectively. Better intestinal absorption, oral bioavailability and cell permeation have been predicted for the test compounds using ADMET protocol available within Discovery Studio 3.0 (DS 3.0). The study thus laid the foundation for the development of better antibacterial agents via a combination approach and safe agricultural fungicides.

In silico analysis and overexpression of chitinase class IV gene in Brassica juncea improves resistance against Alternaria brassicae

Indian mustard (Brassica juncea), an elite oilseed crop in the Brassicae family is challenged by various biotic stresses. Alternaria leaf blight is the most devastating disease in B. juncea caused by Alternaria brassicae. Hence engineering B. juncea plants for improved Alternaria disease resistance becomes imperative. Plant chitinases are key players in the plant immune system that offers successful protection to fungal infections over a broad spectrum of diseases. Chitinase class IV gene was isolated and functionally characterized in Indian mustard (Brassica juncea). Based on phylogenetic analysis, Bj chitinase class IV gene was closely related to Brassica napus followed by other Brassica species. To understand the defensive role of this gene, it was cloned in (Pet28a+) vector and expressed in the prokaryotic expression host Escherichia coli BL21. The purified protein showed antifungal activity against A. brassicae. To further discern its role in disease resistance, B. juncea transgenic lines were raised by overexpressing Bj chitinase IV gene and further confirmed by PCR and southern blotting. In addition, independent B. juncea transgenic lines with high transcript levels of chitinase class IV gene were selected for disease assays against A. brassicae infection. Pathological studies showed that the rate of A. brassicae lesions development and size was significantly reduced in the Bj chitinase transgenic lines compared with non transgenic plants. Interestingly, Bj chitinase transgenic lines showed increased activity of ROS scavenging enzymes when compared to control plants. Collectively our results suggest that overexpression of Bj chitinase IV gene in B. juncea improves resistance against Alternaria leaf blight.