Metabolomic profiling of virulent and non-virulent Beauveria bassiana strains: insights into the pathogenicity of Tetranychus truncatus.

In this study, the pathogenicity of local Beauveria bassiana strains was elucidated using molecular and metabolomics methodologies. Molecular verification of the B. bassiana-specific chitinase gene was achieved via phylogenetic analysis of the Bbchit1 region. Subsequent metabolomic analyses employing UPLC-Q-TOF-MS revealed a different number of non-volatile metabolite profiles among the six B. bassiana strains. Bb6 produced the most non-volatile compounds (17) out of a total of 18, followed by Bb15 (16) and Bb12 (15). Similarly, Bb5, Bb8, and Bb21, three non-virulent B. bassiana strains, produced 13, 14, and 14 metabolites, respectively. But unique secondary metabolites like bassianolide and beauvericin, pivotal for virulence and mite management, were exclusively found in the virulent strains (Bb6, Bb12, and Bb15) of B. bassiana. The distinctive non-volatile metabolomic profiles of these strains underscore their pathogenicity against Tetranychus truncatus, suggesting their promise in bio-control applications.

Novel indazolylchromones: synthesis, fungicidal evaluation, molecular docking and aquatic toxicity prediction.

Fungal diseases cause substantial loss to agricultural crops, affecting both quantities and quality. Although several methods are used for preventing disease incidence, fungicides remain crucial for higher yields and better quality. But in the past, the efficacy of several fungicides has decreased due to increased cases of fungicide resistant. In our pursuit of new effective fungicides, we synthesised a series of twenty 2-Indazol-1-yl-chromen-4-one derivatives (6a- 6t). The characterization of synthesized compounds was performed by several spectroscopic methods including Infrared, Nuclear Magnetic Resonance (1H and 13C) and HRMS. Out of 20 synthesised compounds, 19 (6b- 6t) were found to be novel. All synthesised indazolylchromones showed very good antifungal activity against Sclerotium rolfsii and Fusarium oxysporum. Among the tested compounds, 6t and 6f exhibited very good fungicidal activity against S. rolfsii with an ED50 of 10.10 mg L-1 and 16.18 mg L-1, respectively. In case of Fusarium oxysporum compound 6f displayed good' activity with an ED50 value of 27.82 mg L-1. Molecular docking study was done to predict the binding sites of most active compounds, 6t and 6f with Cytochrome P450 14alpha -sterol demethylase (CYP51) enzyme using molsoft software. The acute toxicity predictions the of synthesized compounds for fish (LC50,96 Hr), daphnid (LC50, 48 Hr) and green algae (EC50, 96Hr) and the chronic toxicity predictions (ChV) were assessed using Ecological Structure Activity Relationship (ECOSAR) model. As per ECOSAR prediction, all the chemicals are inside AD and not missing predictions.

Characterizing genetic diversity of Sclerotium rolfsii isolates by biomapping of mycelial compatibility groupings and multilocus sequence analysis.

Genetic diversity in Sclerotium rolfsii is useful for understanding its population structure, identifying different mycelial compatibility groups (MCGs), and developing targeted strategies for disease management in affected crops. In our study, a comprehensive genetic analysis was conducted on 50 isolates of S. rolfsii, collected from various geographic regions and host plants. Two specific genes, TEF1α and RPB2, were utilized to assess the genetic diversity and relationships among these isolates. Notably, out of 1225 pairings examined, only 154 exhibited a compatible reaction, while the majority displayed antagonistic reactions, resulting in the formation of a barrier zone. The isolates were grouped into 10 distinct MCGs. These MCGs were further characterized using genetic sequencing. TEF1α sequences distinguished the isolates into 17 distinct clusters, and RPB2 sequences classified them into 20 clusters. Some MCGs shared identical gene sequences within each gene, while others exhibited unique sequences. Intriguingly, when both TEF1α and RPB2 sequences were combined, all 10 MCGs were effectively differentiated, even those that appeared identical with single-gene analysis. This combined approach provided a comprehensive understanding of the genetic diversity and relationships among the S. rolfsii isolates, allowing for precise discrimination between different MCGs. The results shed light on the population structure and genetic variability within this plant pathogenic fungus, providing valuable insights for disease management and control strategies. This study highlights the significance of comprehending the varied virulence characteristics within S. rolfsii isolates, categorizing them into specific virulence groups based on disease severity index (DSI) values. The association with MCGs provides additional insights into the genetic underpinnings of virulence in this pathogen. Furthermore, the identification of geographical patterns in virulence implies the influence of region-specific factors, with potential implications for disease control and crop protection strategies.Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Author 1 Given name: [G. M. Sandeep] Last name [Kumar]. Author 2 Given name: [Praveen Kumar] Last name [Singh]. Also, kindly confirm the details in the metadata are correct.I confirm that the given names are accurate and presented in the correct sequence.

Molecular characterization of Indian races of Fusarium oxysporum f. sp. lentis (Fol) based on secreted in Xylem (SIX) effector genes and development of a SIX11 gene-based molecular marker for specific detection of Fol.

Fusarium wilt of lentil caused by Fusarium oxysporum f. sp. lentis (Fol) is a destructive pathogen limiting lentil production in India. In the present study, Secreted in Xylem (SIX) effectors genes were explored in Indian races of Fol and also a diagnostic tool for reliable detection of the disease was developed. Four SIX effectors genes, SIX11, SIX13, SIX6 and SIX2 were identified in 12 isolates of Fol belonging to seven races. SIX11 was present in all the races while SIX 13 was absent in race 6 and SIX6 was present only in race 4. The phylogenetic analysis revealed the conserved nature of the SIX genes within the forma specialis and showed sequence homology with F. oxysporum f. sp. pisi. The presence of three effectors, SIX11, SIX13 and SIX6 in race 4 correlates with high disease incidence in lentil germplasms. The in-silico characterization revealed the presence of signal peptide and localization of the effectors. Further SIX11 effector gene present in all the isolates was used to develop Fol-specific molecular marker for accurate detection. The marker developed could differentiate F. oxysporum f. sp. lycopersici, F. solani, F. oxysporum, Rhizoctonia solani and Sclerotium rolfsii and had a detection limit of 0.01ng µL- 1. The effector-based marker detection helps in the unambiguous detection of the pathogen under field conditions.

Expression profiling and characterization of key RGA involved in lentil Fusarium wilt Race 5 resistance.

Fusarium wilt is a major threat to lentil production in India and worldwide. The presence of evolving virulent races has imposed the necessity of reliable management practices including breeding for resistance using unexplored germplasms. The magnitude of resistance by the plant is determined by rapid recognition of the pathogen and induction of defence genes. Resistance gene analogues have been key factors involved in the recognition and induction of defence response. In the present study, the expression of key RGA previously cloned was determined in three resistant accessions (L65, L83 and L90) and a susceptible accession (L27). The expression was assessed via qPCR at 24, 48 and 72 hpi against virulent race5 (CG-5). All the RGAs differentially transcribed in resistant and susceptible accession showed temporal variation. RGA Lc2, Lc8, Ln1 and Lo6 produced cDNA signals during early infection (24 hpi) predicting its involvement in recognition. LoRGA6 showed significant upregulation in L65 and L83 while downregulating in L27 and the full length of LoRGA6 loci was isolated by 5' and 3' RACE PCR. In-silico characterization revealed LoRGA6 loci code for 912 amino acids long polypeptide with a TIR motif at the N terminal and eight LRR motifs at the C terminal. The tertiary structure revealed a concave pocket-like structure at the LRR domain potentially involved in pathogen effectors interaction. The loci have ADP binding domain and ATPase activity. This has further paved the path for functional analysis of the loci by VIGS to understand the molecular mechanism of resistance.

Essential oil-grafted copper nanoparticles as a potential next-generation fungicide for holistic disease management in maize.

Sustainable food production is necessary to meet the demand of the incessantly growing human population. Phytopathogens pose a major constraint in food production, and the use of conventional fungicides to manage them is under the purview of criticism due to their numerous setbacks. In the present study, essential oil-grafted copper nanoparticles (EGC) were generated, characterized, and evaluated against the maize fungal pathogens, viz., Bipolaris maydis, Rhizoctonia solani f. sp. sasakii, Macrophomina phaseolina, Fusarium verticillioides, and Sclerotium rolfsii. The ED50 for the fungi under study ranged from 43 to 56 µg ml-1, and a significant inhibition was observed at a low dose of 20 µg ml-1 under in vitro conditions. Under net house conditions, seed treatment + foliar spray at 250 and 500 mg L-1 of EGC performed remarkably against maydis leaf blight (MLB), with reduced percent disease index (PDI) by 27.116 and 25.292%, respectively, in two Kharif seasons (May-Sep, 2021, 2022). The activity of enzymatic antioxidants, viz., ß-1, 3-glucanase, PAL, POX, and PPO, and a non-enzymatic antioxidant (total phenolics) was increased in treated maize plants, indicating host defense was triggered. The optimum concentrations of EGC (250 mg L-1 and 500 mg L-1) exhibited improved physiological characteristics such as photosynthetic activity, shoot biomass, plant height, germination percentage, vigor index, and root system traits. However, higher concentrations of 1,000 mg L-1 rendered phytotoxicity, reducing growth, biomass, and copper bioaccumulation to high toxic levels, mainly in the foliar-sprayed maize leaves. In addition, EGC and copper nanoparticles (CuNPs) at 1,000 mg L-1 reduced the absorption and concentration of manganese and zinc indicating a negative correlation between Cu and Mn/Zn. Our study proposes that the CuNPs combined with EO (Clove oil) exhibit astounding synergistic efficacy against maize fungal pathogens and optimized concentrations can be used as an alternative to commercial fungicides without any serious impact on environmental health.

Resistance screening and in silico characterization of cloned novel RGA from multi-race resistant lentil germplasm against Fusarium wilt (Fusarium oxysporum f. sp. lentis).

Fusarium wilt caused by Fusarium oxysporum f. sp. lentis (Fol) is the most devastating disease of lentil present worldwide. Identification of multi-race fusarium wilt resistance genes and their incorporation into existing cultivars will help to reduce yield losses. In the present study, 100 lentil germplasms belonging to seven lentil species were screened against seven prevalent races of Fol, and accessions IC201561 (Lens culinaris subsp. culinaris), EC714243 (L. c. subsp. odemensis), and EC718238 (L. nigricans) were identified as resistant. The typical R gene codes for the nucleotide-binding site and leucine-rich repeats (NBS-LRR) at the C terminal are linked to either the Toll/interleukin 1-like receptor (TIR) or coiled coil (CC) at the N terminal. In the present study, degenerate primers, designed from the NBS region amplifying the P-loop to the GLPLA motif, isolated forty-five resistance gene analogues (RGAs) from identified resistant accessions. The sequence alignment identified both classes of RGAs, TIR and non-TIR, based on the presence of aspartate (D) and tryptophan (W) at the end of the kinase motif, respectively. The phylogenetic analysis grouped the RGAs into six classes, from LRGA1 to LRGA6, which determined the diversity of the RGAs present in the host. Grouping of the RGAs identified from Lens nigricans, LnRGA 2, 9, 13 with I2 revealed the structural similarity with the fusarium resistance gene. The similarity index ranged from 27.85% to 86.98% among the RGAs and from 26.83% to 49.41% among the known R genes, I2, Gpa2, M, and L6. The active binding sites present along the conserved motifs grouped the RGAs into 13 groups. ADP/ATP, being the potential ligand, determines the ATP binding and ATP hydrolysis activity of the RGAs. The isolated RGAs can be used to develop markers linked to the functional R gene. Furthermore, expression analysis and full-length gene isolation pave the path to identifying the molecular mechanism involved in resistance.

Metabolomic profiling and its association with the bio-efficacy of Aspergillus niger strain against Fusarium wilt of guava.

Bio-control agents are the best alternative to chemicals for the successful management of plant diseases. The fungus Aspergillus niger is known to produce diverse metabolites with antifungal activity, attracting researchers to exploit it as a bio-control agent for plant disease control. In the present study, 11 A. niger strains were isolated and screened for their antagonism against the guava wilt pathogen under in vitro and in planta conditions. Strains were identified morphologically and molecularly by sequencing the internal transcribed spacer (ITS), ß-tubulin, and calmodulin genes. The strains were evaluated through dual culture, volatile, and non-volatile methods under an in vitro study. AN-11, AN-6, and AN-2 inhibited the test pathogen Fusarium oxysporum f. sp. psidii (FOP) at 67.16%, 64.01%, and 60.48%, respectively. An in planta study was conducted under greenhouse conditions with 6 months old air-layered guava plants (var. Allahabad Safeda) by pre- and post-inoculation of FOP. The AN-11 strain was found to be effective under both pre- and post-inoculation trials. Furthermore, gas chromatography-mass spectrometry (GC-MS) analysis was carried out to characterize the volatile compounds of the most potential strain, A. niger. The hexane soluble fraction showed the appearance of characteristic peaks of hexadecenoic acid methyl ester (4.41%), 10-octadecanoic acid methyl ester (3.79%), dodecane (3.21%), undecane (3.19%), gibepyrone A (0.15%), 3-methylundecane (0.36%), and citroflex A (0.38%). The ethyl acetate fraction of the bio-control fungi revealed the occurrence of major antifungal compounds, such as acetic acid ethyl ester (17.32%), benzopyron-4-ol (12.17%), 1,2,6-hexanetriol (7.16%), 2-propenoic acid ethanediyl ester (2.95%), 1-(3-ethyloxiranyl)-ethenone (0.98%), 6-acetyl-8-methoxy dimethyl chromene (0.96%), 4-hexyl-2,5-dihydro dioxo furan acetic acid (0.19%), and octadecanoic acid (1.11%). Furthermore, bio-control abilities could be due to hyper-parasitism, the production of secondary metabolites, and competition for sites and nutrients. Indeed, the results will enrich the existing knowledge of metabolomic information and support perspectives on the bio-control mechanism of A. niger.

Exploring Potent Fungal Isolates from Sanitary Landfill Soil for In Vitro Degradation of Dibutyl Phthalate.

Di-n-butyl phthalate (DBP) is one of the most extensively used plasticizers for providing elasticity to plastics. Being potentially harmful to humans, investigating eco-benign options for its rapid degradation is imperative. Microbe-mediated DBP mineralization is well-recorded, but studies on the pollutant's fungal catabolism remain scarce. Thus, the present investigation was undertaken to exploit the fungal strains from toxic sanitary landfill soil for the degradation of DBP. The most efficient isolate, SDBP4, identified on a molecular basis as Aspergillus flavus, was able to mineralize 99.34% dibutyl phthalate (100 mg L-1) within 15 days of incubation. It was found that the high production of esterases by the fungal strain was responsible for the degradation. The strain also exhibited the highest biomass (1615.33 mg L-1) and total soluble protein (261.73 µg mL-1) production amongst other isolates. The DBP degradation pathway scheme was elucidated with the help of GC-MS-based characterizations that revealed the formation of intermediate metabolites such as benzyl-butyl phthalate (BBP), dimethyl-phthalate (DMP), di-iso-butyl-phthalate (DIBP) and phthalic acid (PA). This is the first report of DBP mineralization assisted with A. flavus, using it as a sole carbon source. SDBP4 will be further formulated to develop an eco-benign product for the bioremediation of DBP-contaminated toxic sanitary landfill soils.

Screening of short-day onions for resistance to Stemphylium leaf blight in the seed-to-bulb stage (stage I) and bulb-to-seed stage (stage II).

Stemphylium leaf blight, caused by Stemphylium vesicarium, is a very important fungal disease in onions since its epidemics are able to affect both the bulb yield and the seed quality. The aim of this study was to screen onion genotypes at stage I (seed to bulb) and further screen the identified resistant and susceptible genotypes at stage II (bulb to seed). One hundred and fifty-seven genotypes were screened against SLB under artificially inoculated field conditions. Results revealed a significant variation among the morphological and biochemical traits studied. Correlation studies revealed a significant and negative correlation between percent disease incidence (PDI), pseudostem width, neck thickness, and dry matter. Fifteen genotypes were identified as moderately resistant, and the rest were categorized as susceptible. Bulbs of the genotypes, identified as moderately resistant, were again screened for resistance in stage II. All the genotypes were categorized as moderately susceptible. Biochemical analysis revealed that total foliar phenol content, pyruvic acid, catalase, and peroxidase increased up to 20 days after inoculation (DAI) and thereafter declined. Protein content was highest in the initial stage and declined at 10, 20, and 30 DAI. The higher biochemical activity was observed in moderately resistant category genotypes compared with the susceptible ones. Correlation analysis showed a highly significant and negative correlation of PDI with total foliar phenol content (TFPC), pyruvic acid, catalase, peroxidase, and protein content. To conclude, it was observed that screening against SLB should be done at both the stages (stage I and Stage II) to identify resistant onion genotypes. Direction selection for genotypes with high dry matter, higher phenols, and enzymes may be an alternative pathway to select genotypes for a robust resistance breeding program.

"Metabolomic diversity of local strains of Beauveria bassiana (Balsamo) Vuillemin and their efficacy against the cassava mite, Tetranychus truncatus Ehara (Acari: Tetranychidae)".

A desirable substitute for chemical pesticides is mycopesticides. In the current investigation, rDNA-ITS (Internal transcribed spacer) and TEF (Transcriptional Elongation Factor) sequencing were used for molecular identification of six Beauveria bassiana strains. Both, leaf discs and potted plant bioassaye were carried out to study their pathogenicity against the cassava mite, Tetranychus truncatus. LC50 and LC90 values of potential B. bassiana strains were estimated. We also discovered a correlation between intraspecific B. bassiana strains pathogenicity and comprehensive metabolome profiles. Bb5, Bb6, Bb8, Bb12, Bb15, and Bb21 strains were identified as B. bassiana by sequencing of rDNA-ITS and TEF segments and sequence comparison to NCBI (National Center for Biotechnology Information) GenBank. Out of the six strains tested for pathogenicity, Bb6, Bb12, and Bb15 strains outperformed against T. truncatus with LC50 values 1.4×106, 1.7×106, and 1.4×106 and with a LC90 values 7.3×107, 1.4×108, and 4.2×108 conidia/ml, respectively, at 3 days after inoculation and were considered as potential strains for effective mite control. Later, Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the above six B. bassiana strains was done on secondary metabolites extracted with ethyl acetate revealed that the potential B. bassiana strains (Bb6, Bb12, and Bb15) have higher levels of acaricidal such as Bis(dimethylethyl)-phenol: Bb6 (5.79%), Bb12 (6.15%), and Bb15 (4.69%). Besides, insecticidal (n-Hexadecanoic acid), and insect innate immunity overcoming compound (Nonadecene) were also identified; therefore, the synergistic effect of these compounds might lead toa higher pathogenicity of B. bassiana against T. truncatus. Further, these compounds also exhibited two clusters, which separate the potential and non-potential strains in the dendrogram of Thin Layer Chromatography. These results clearly demonstrated the potentiality of the B. bassiana strains against T. truncatus due to the occurrence of their bioactive volatile metabolome.

Performance appraisal of Trichoderma viride based novel tablet and powder formulations for management of Fusarium wilt disease in chickpea.

In developing a Trichoderma viride-based biocontrol program for Fusarium wilt disease in chickpea, the choice of the quality formulation is imperative. In the present study, two types of formulations i.e. powder for seed treatment (TvP) and tablet for direct application (TvT), employing T. viride as the biocontrol agent, were evaluated for their ability to control chickpea wilt under field conditions at three dosages i.e. recommended (RD), double of recommended (DD) and half of recommended (1/2 RD). A screening study for the antagonistic fungi strains based on volatile and non-volatile bioassays revealed that T. viride ITCC 7764 has the most potential among the five strains tested (ITCC 6889, ITCC 7204, ITCC 7764, ITCC 7847, ITCC 8276), which was then used to develop the TvP and TvT formulations. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of volatile organic compounds (VOCs) of T. viride strain confirmed the highest abundance of compositions comprising octan-3-one (13.92%), 3-octanol (10.57%), and 1-octen-3-ol (9.40%) in the most potential T. viride 7764. Further Physico-chemical characterization by standard Collaborative International Pesticides Analytical Council (CIPAC) methods revealed the optimized TvP formulation to be free flowing at pH 6.50, with a density of 0.732 g cm-3. The TvT formulation showed a pH value of 7.16 and density of 0.0017 g cm-3 for a complete disintegration time of 22.5 min. The biocontrol potential of TvP formulation was found to be superior to that of TvT formulation in terms of both seed germination and wilt incidence in chickpea under field conditions. However, both the developed formulations (TvP and TvT) expressed greater bioefficacy compared to the synthetic fungicide (Carbendazim 50% WP) and the conventional talc-based formulation. Further research should be carried out on the compatibility of the developed products with other agrochemicals of synthetic or natural origin to develop an integrated disease management (IDM) schedule in chickpea.

"Multilocus sequence analysis for population diversity of indigenous entomopathogenic fungus Beauveria bassiana and its bio-efficacy against the cassava mite, Tetranychus truncatus Ehara (Acari: Tetranychidae)".

Beauveria bassiana is an entomopathogenic fungus that causes the white muscadine disease in insects. The majority of entomopathogenic fungi are soil and insect borne, 15 soil samples were collected from seven different locations during 2021, from January to December. Similarly, during 2022, March to December, 15 fungus-infected insect specimens were collected from five different locations hence soil and insect samples from various ecosystems were collected. As a result, 30 B. bassiana isolates from 11 different geographical areas were identified using morphological characteristics and multilocus sequence data in this investigation. The taxonomical positions of the isolates were determined using morphological characteristics and phylogenetic inferences based on three loci (Internal Transcribed Sequence, Elongation Factor-1α, and B. bassiana chitinase 1). In phylogenetic analysis of B. bassiana, the Maximum Likelihood analytical method produced distinct tree topology when compared to Neighbor-joining and minimum evolution. Three isolates viz., Bb3, Bb7 and Bb20 were found closely linked with reference isolate (KTU-24) and other showed the higher population diversity among them. The genetic distances of 30 B. bassiana isolates revealed that 15 were not closely related (D varied from 0.003 to 0.036). The pathogenicity of B. bassiana isolates from various hosts along with one commercial formulation (Beveroz) was assessed against Tetranychus truncatus under in vitro conditions by a completely randomized design (CRD) experiment. The same experiment was repeated thrice to confirm the pathogenicity of B. bassiana against T. truncatus. Later, the collected T. truncatus mortality data was converted into corrected mortality by using the Abbott formula and the values were examined using analysis of variance (ANOVA) in SPSS 23.0 software. Duncan's Multiple Comparison Test was also done to compare the percentage mortality rates among the 30 B. bassiana isolates. The recorded results showed that the Bb6, Bb15 and Bb12 isolates caused significantly higher mortality of T. truncatus, i.e., 97.73, 96.73 and 94.50% respectively, than the other isolates. This study showed the relativeness among the B. bassiana isolates and establishes their bio-efficacy against T. truncatus, which further can be used for commercialization as bio-pesticide.

Bioefficacy evaluation of ferrocenyl chalcones against Meloidogyne incognita and Sclerotium rolfsii infestation in tomato.

The present study reports, bioefficacy evaluation of effective compounds against Meloidogyne incognita and Sclerotium rolfsii in pot cultured tomato. The identified five most effective compounds, i.e. (2E)-1-(4-Methylphenyl)-3-ferrocenyl-prop-2-en-1-one (6g), (2E)-1-(4-Methoxyphenyl)-3-ferrocenyl-prop-2-en-1-one (6h), (2E)-1-(3-Bromophenyl)-3-ferrocenyl-prop-2-en-1-one (6j), (2E)-1-(2,4-Dichlorophenyl)-3-ferrocenyl-prop-2-en-1-one (6k) and (2E)-1-(3,5-Dichloro-2-hydroxyphenyl)-3-ferrocenyl-prop-2-en-1-one (6p) along with Carbofuran 3G as positive control were tested at 20, 40 and 80 ppm by soil drenching and root dipping methods. The study revealed that all plant growth parameters were positively influenced by these compounds. The presence of an electron releasing group positively influenced the efficacy, and the activity was highest in compounds 6g and 6h at 80 ppm. Based on in vitro results against S. rolfsii, (2E)-1-Ferrocenyl-3-(4-bromophenyl)-prop-2-en-1-one (3b), (2E)-1-Ferrocenyl-3-(2,6-dichlorophenyl)-prop-2-en-1-one (3o) and (2E)-1-(5-Chloro-2-hydroxyphenyl)-3-ferrocenyl-prop-2-en-1-one (6o) along with Tebuconazole 25.9% EC and Hexaconazole 5% SC as positive control were evaluated. The shoot length was found to be highest (24.50 cm) in plants treated with 3b followed by 3o and 6o at 1000 ppm. The percent disease incidence was significantly decreased as compared to control. The percent disease incidence was found to be minimum in plants treated with 3b at 1000 ppm. However, root dipping was not as effective as soil drenching. Therefore, ferrocenyl chalcone derivatives proved to be of great fungicidal and nematicidal potential opening new opportunities for expanding their effectiveness as new pest control agents.

Diversity analysis of different Diaporthe (Phomopsis) species and development of molecular marker to identify quarantine important species Phomopsis phaseolorum.

The genus Diaporthe Nitschke (Phomopsis Sacc. & Harter) infect various agricultural and horticultural important crops and cause diseases such as damping off, leaf spots, blights, canker, dieback, wilt, root and fruit rots. P. vexans, P. helianthi and P. phaseolorum are the important species within genus causing huge yield and economic loss. Being primarily seed borne it also hinders import and export of germplasm and seeds. Therefore, extensive characterization is required to diagnose and manage the disease. Seventeen isolates collected from ITCC, IARI and ICAR-NBPGR belonging to eight species were morphological and molecularly characterized and diversity was analyzed. Several morphological and cultural characters were studied and analyzed. Due to lack of sufficient morphological variation to identify/differentiate species, molecular characterization using house-keeping genes, internal transcriber spacer (ITS) was carried out. ITS produced amplicon of ~ 600 bp in the isolates of Phomopsis and phylogenetic tree obtained revealed that isolates of a species belonging same geographic region had more sequence similarity than isolates belonging to different geographic regions this might be due to population adaption under varied environments. Development of EF-1alpha-based marker specific to P. phaseolorum helps in easily detection of pathogen in quarantine stations. In addition, species of Phomopsis were previously named based on host association which has led to misidentification and proliferation of species. Cross pathogenicity of isolates on three important hosts, brinjal, soybean and chilli revealed its broad host range and naming only basis of host association is unjustified.

Identification, Characterization, and Evaluation of Nematophagous Fungal Species of Arthrobotrys and Tolypocladium for the Management of Meloidogyne incognita.

Root-knot nematodes belonging to the genus Meloidogyne are agriculturally important pests, and biocontrol strategies offer safer alternatives for their management. In the present study, two fungal species from Indian soils were identified as Arthrobotrys thaumasia and Tolypocladium cylindrosporum based on morphological characteristics and further confirmed using molecular markers. In vitro evaluation of A. thaumasia against M. incognita and Caenorhabditis elegans showed 82 and 73% parasitism, respectively, whereas T. cylindrosporum gave 65.2 and 57.7% parasitism, respectively. Similarly, culture filtrates of A. thaumasia caused 57.7 and 53.7% mortality of M. incognita and C. elegans, respectively, whereas T. cylindrosporum caused higher mortality of 87.3 and 64%, respectively. Besides, greenhouse evaluation of both fungi against M. incognita infecting tomato significantly reduced nematode disease burden reflecting parasitic success measured as the total number of galls, egg masses, eggs per egg mass, and derived nematode multiplication factor. Application of A. thaumasia and T. cylindrosporum reduced nematode multiplication factor by 80 and 95%, respectively, compared with control. General metabolite profiling of tested fungi using gas chromatography-mass spectrometry and ultra-performance liquid chromatography-quadrupole/time of flight mass spectrometry reported for the first time here showed presence of various volatile and non-volatile compounds with nematicidal activity, viz., trimethyl-heptadiene, methyl-hexadecanol, dodecadienal, decane, terpendole E, dodecane, acetamido-6-anthraquinone, and hexadecanol. Also, other compounds such as undecane, dibutyl-disulfide, octadecenal, paganin, talathermophilin, dactylarin, tolypyridone A, tolypyridone B, pyridoxatin, and destruxin were identified, reported in the literature to possess antibacterial, antifungal, and insecticidal properties. This is the first report of the occurrence of both fungi from India and pioneer demonstration of T. cylindrosporum for root-knot nematode management.

Microwave assisted synthesis, characterization and biological activities of ferrocenyl chalcones and their QSAR analysis: Part II.

A series of ferrocenyl chalcones using acetylferrocene, with ferrocenyl group at the keto carbonyl group, and different aldehydes were synthesized and their bioefficacy evaluation was done against Sclerotium rolfsii, Alternaria solani and Meloidogyne incognita. In continuation of our quest for potent crop protection products, in the present study, a series of 18 substituted ferrocenyl chalcones were synthesized in which ferrocenyl group was attached to the aldehyde moiety, using ferrocenecarboxyaldehyde and different acetophenones by microwave method (MM) and conventional method (CM) [cf: MM 1 to 5 min; CM 12-40 h] and characterized by various techniques viz. IR, LC-HRMS, 1H-NMR and 13C-NMR. In vitro fungicidal activity showed that compound, (2E)-1-(5-Chloro-2-hydroxyphenyl)-3-ferrocenyl-prop-2-en-1-one (34) (ED50 = 21.50 mg L-1) was found to be most active against S. rolfsii and compound, (2E)-1-(4-Bromophenyl)-3-ferrocenyl-prop-2-en-1-one (21) (ED50 = 31.14 mg L-1) showed highest activity against A. solani. As regards nematicidal activity, compound (2E)-1-(3-Bromophenyl)-3-ferrocenyl-prop-2-en-1-one (29) was more potent with LC50 values of 11.95, 8.07 and 4.34 mg L-1 at 24, 48 and 72 h, respectively. QSAR study revealed that MLR for S. rolfsii (r 2 = 0.9834, q 2= 0.8975) and A. solani (r 2 = 0.9807, q 2= 0.8713) and PLS for M. incognita (r 2 = 0.9023, q 2= 0.7818) were the best models.

First report of stem smut caused by Tranzscheliella hypodytes (Schltdl.) Vanky & McKenzie on Leymus secalinus (Georgi) Tzvelev in India.

Leymus secalinus (Blue wild rye) is a perennial grass species distributed in Leh-Ladakh region of India. Culms are usually solitary, 20-100 cm tall, 2-5-noded, smooth and glabrous. It is found on mountain slopes, rocky, stony and pebbled soils, grassy places, river banks, sandy and alkaline soils. It is one of the dominant species of the region and is mostly used for forage and grazing. L. secalinus plants with blackish-brown powdery spore mass/sori on the culm was observed in Leh region of Jammu and Kashmir, India during a wheat germplasm exploration (to collect wild relatives, land races, cultivars etc. of cultivated wheat) in September, 2018. Initially, sori were covered by the leaf sheath and at later stage more or less exposed with the absence of peridium. Infected culms and leaves are stunted, while inflorescences are abortive. Spores are globose, sub-globose to ovoid, blackish-brown in color, 3-5 x 4-4.5 µm in size, wall 0.5 µm thick and smooth. The fungus was identified as Tranzscheliella hypodytes (Schltdl.) Vanky & McKenzie based on morphological characteristics (Li et al. 2017; Vanky 2012, 2003; Vanky and McKenzie 2002). The ustilospore germination was not observed on potato dextrose agar during the lab experiment. In some cases, ustilospore germination is recorded in other smut fungi which shows considerable variation according to media and conditions used (Ingold, 1983). Moreover, there is no report available for the artificial culture of T. hypodytes. Identity was confirmed using the internal transcribed spacer (ITS) region of ribosomal DNA, amplified with universal primers ITS1 which was then sequenced. NCBI-BLAST showed 99.28% similarity with the ITS sequences of Tranzscheliella hypodytes (GenBank accession no. MH855460.1). The sequence was deposited in NCBI GenBank with accession number (MN108147). Pathogenecity test was conducted in glasshouse wherein smut infected samples of L. secalinus collected from Leh were made into small pieces and mixed with the sterilized soil filled in three pots before sowing. In addition, seeds were also mixed with smut spores before sowing. Inoculated pots after sowing were kept at 16±20C temperature and a relative humidity of 70% in a glass house. Control pots were maintained without inoculum. Inoculated and control pots were placed in separate chambers of the same glasshouse. Seventy days after sowing, inoculated plants were symptomatic, matching those described above and produced no seed. Pathogen reisolated from infected plants was identical in all respects to the isolate used to inoculate the plants, which confirmed the Koch's postulates. Control plants remained healthy and produced seed. Leh region is covered by sparse vegetation due to its topography and cold desert climate (cold, dry winter and warm, dry summer with very low precipitation, occuring in temperate region). Stem smut in L. secalinus can have significant negative impact on the fodder and grazing situation of the region. To our knowledge, this is the first report of stem smut caused by T. hypodytes on L. secalinus in India. A voucher specimen of the fungus was deposited at Herbarium Cryptogamae Indiae Orientialis (HCIO) (52182), ICAR-Indian Agricultural Research Institute, New Delhi.

First Report of Stem Rot Caused by Athelia rolfsii on Curry Leaf Tree (Murraya koenigii) in Tripura, India.

Murraya koenigii is an important medicinal plant of India and commonly known as curry leaf tree grown in tropical and subtropical regions. The leaves of curry tree are used as a herb due to the presence of following important active constituent bismahanine, murrayanine, murrayafoline-A, bi-koeniquinone-A, murrayazolidine etc. (Jain et al. 2017). During mid-July 2019, stem rot disease symptoms were observed on curry leaf trees at the College of Agriculture, Lembucherra, Tripura (India). The disease symptoms consisted of rotting, wilting and blighting with disease incidence ranging from 8 to 10%. Initially, infected plants gradually withered and white mycelia mats appeared on the surface of the lower stem at the soil line. Infected stem samples were collected and surface was sterilized with 0.25% sodium hypochlorite for 1 min, washed thrice with sterilized distilled water and placed in Petri plates containing 2% water agar. After three days of incubation at 26°C, hyphae produced from plant bits were transferred into Petri plates containing potato dextrose agar. Ten isolates were collected from the diseases samples. Pure cultures were obtained as abundant, aerial and white mycelia with round to irregular sclerotia of 0.8 to 1.5 mm in diam. The sclerotia were initially white in color but later turned into brown color. The pathogen was identified as Athelia rolfsii based on morphology (Aycock 1966). To confirm the identification, the genomic DNA was extracted from a mycelia mat of the isolates using ZR fungal/Bacterial DNA miniprep kit (Irvine, CA) and the internal transcribed spacer (ITS) region of rDNA was amplified using the universal primers, ITS1 and ITS4 (White et al. 1990). A 550 bp PCR product was sequenced and showed 99% similarity with Athelia rolfsii isolate (GenBank accession MH854711).The generated sequence was submitted to GenBank (Accession MT535585). After identification of the pathogen a pot experiment was conducted to confirm the pathogenicity. Earthen pots (29 cm. diam.) were filled with sterilized soil and kept in a green house. Ten curry leaf plants (50 days old) were grown from seed in the separate pot were inoculated with 15-day old mycelia mats prepared in potato dextrose broth. The stem of each curry plant were artificially injured with the help of sterile blade and wrapped with moistened sterilized cotton containing the mycelial mat. Five curry leaf plants artificially injured and inoculated with sterilized distilled water were used as control. The Earthen pots with plants were individually covered with plastic bags and kept in the green house at 26°C for approximately 15 days. The inoculated plants started showing symptoms of stem rot six days after inoculation and started drying onward. The symptoms of stem rot on the inoculated plants were similar to those observed in the field. The fungus was re-isolated from the inoculated plants and A. rolfsii identification was confirmed based on morphology. No symptoms were observed on the control plants. The obtained culture was deposited in the Indian Type Culture Collection, Division of Plant Pathology, ICAR - Indian Agricultural Research Institute, New Delhi, India (ITC-8666). To the best of our knowledge this is the first report of stem rot disease of curry leaf plant caused by A. rolfsii in India and worldwide. Due to medicinal, flavour and aroma properties, it is regularly used in India. Curry leaf plant is regularly used as a medical herb in India and therefore this disease poses a significant risk to production.

Microwave Assisted Synthesis, Characterization and Biological Activities of Ferrocenyl Chalcones and Their QSAR Analysis.

A new microwave method (MM) has been developed for the synthesis of a series of 16 substituted ferrocenyl chalcones using acetylferrocene (1) with different aldehydes (2a-2p) and comparing it with conventional method (CM). The synthesized compounds were characterized by various spectroscopic techniques viz IR, HR-MS, 1H NMR, and 13C NMR. The time required for completion of reaction in MM varied from 1 to 5 min as compared to CM which required 10-40 h. All the synthesized compounds were screened for antifungal activity against Sclerotium rolfsii and Alternaria solani. In vitro fungicidal activity revealed that compound 3o (ED50 = 23.24 mg L-1) was found to be most active against S. rolfsii. But in case of A. solani, compound 3c (ED50 = 29.9 mg L-1) showed highest activity. The nematicidal activity revealed that the compound 3b was more potent with LC50 values of 10.67, 7.30, and 4.55 ppm at 24, 48, and 72 h, respectively. 2D-Quantitative Structural Activity Relationship (2D-QSAR) analysis of these ferrocenyl chalcones was carried out by developing three different models namely Partial Least Squares (PLS, Model 1), Multiple Linear Regression (MLR, Model 2) and Principal Component Regression (PCR, Model 3). Statistical significance and predictive ability of these models were assessed by internal and external validation and also verified by leave one-out cross-validation. QSAR study revealed that MLR for S. rolfsii (r 2 = 0.999, q 2 = 0.996), PLS for A. solani (r 2 = 0.934, q 2 = 0.749) and PCR for M. incognita (r 2 = 0.878, q 2 = 0.772) were the best model. The physico-chemical parameters were calculated using VLife MDS 4.6 software. QSAR study could be employed for structure optimization to achieve better activity.