Metagenomic Approach Deciphers the Role of Community Composition of Mycobiome Structured by Bacillus velezensis VB7 and Trichoderma koningiopsis TK in Tomato Rhizosphere to Suppress Root-Knot Nematode Infecting Tomato.

The soil microbiome is crucial for maintaining the sustainability of the agricultural environment. Concerning the role of diverse mycobiomes and their abundance toward the suppression of root-knot nematode (RKN) infection in vegetable crops, our understanding is unclear. To unveil this issue, we examined the fungal microbiome in tomato rhizosphere augmented with bioagents challenged against RKN at taxonomic and functional levels. Composition of the mycobiome in tomato rhizosphere treated with Bacillus velezensis VB7 and Trichoderma koningiopsis TK differed significantly from the infected tomato rhizosphere. The abundance and diversity of fungal species, however, were significantly higher in the combined treatments of bioagents than for individual treatments. Fungal microbiome diversity was negatively correlated in the RKN-associated soil. Network analysis of the fungal biome indicated a larger and complex network of fungal biome diversity in bioagent-treated soil than in nematode-associated tomato rhizosphere. The diversity index represented by that challenging the RKN by drenching with consortia of B. velezensis VB7 and T. koningiopsis TK, or applying them individually, constituted the maximum abundance and richness of the mycobiome compared to the untreated control. Thus, the increased diverse nature and relative abundance of the mycobiome in tomato rhizosphere was mediated through the application of either T. koningiopsis TK or B. velezensis VB7, individually or as a consortium comprising both fungal and bacterial antagonists, which facilitated engineering the community composition of fungal bioagents. This in turn inhibited the infestation of RKN in tomato. It would be interesting to explore further the possibility of combined applications of B. velezensis VB7 and T. koningiopsis TK to manage root-knot nematodes as an integrated approach for managing plant parasitic nematodes at the field level.

Activity of volatiles induced by microbes and natural plants stifled the growth of Pythium aphanidermatum - the damping off in Tomato.

BACKGROUND: Volatilomes from natural plants and microbes imparts diverse antifungal properties to suppress the growth of plant pathogens and therefore can be a suitable alternative of chemical fungicides. The present experiment was to study effect of volatiles produced by natural plants and microbes on the fungal growth of Pythium aphanidermatum, which is a tomato seedling pathogen. RESULTS: Isolate of P. aphanidermatum, causing damping off in tomato were isolated and incubated at 25 ± 2 °C. The isolate was tested for the anti-oomycetes activities of volatiles in vitro. The volatiles produced by the leaves of Mentha spicata and Cymbopogon citratus showed the maximum inhibitory effect of 45.56 and 24.70 percent, respectively on the mycelial growth of P. aphanidermatum, whereas, the pathogen was not inhibited on exposure to the volatiles of macro-basidiomycetes fungi. The volatiles of T. asperellum showed the maximum inhibitory effect of 69.26 percent against P. aphanidermatum. The study also included the identification of Volatile Organic Compounds (VOCs) involved in the suppression of pathogens by Headspace Gas Chromatography Mass Spectrometry (HS GCMS). The results revealed the production of carvone by the leaves of M. spicata; citronellol and geraniol by C. citratus; isopentyl alcohol and limonene by T. asperellum with increased peak area percentage and these compounds possessed antifungal properties. The vaporous action of isopentyl alcohol completely suppressed the mycelial growth of P. aphanidermatum, which is highly correlated to the T. asperellum extract on pathogenic growth. While the compounds, carvone, and citronellol showed the maximum inhibitory effect of 89.02 and 85.49 percent, respectively when used at 500 ppm and also altered the sporulation behavior of P. aphanidermatum. CONCLUSION: Results showed that volatiles of M. spicata and T. asperellum have anti-oomycetes action on pathogenic growth leading to a distortion of sporulation of P. aphanidermatum. High antifungal properties make VOCs suitable for incorporation as a new integrated plant disease management programs.

Antagonistic Bacteria Bacillus velezensis VB7 Possess Nematicidal Action and Induce an Immune Response to Suppress the Infection of Root-Knot Nematode (RKN) in Tomato.

Meloidogyne incognita, the root-knot nematode (RKN), a devastating plant parasitic nematode, causes considerable damage to agricultural crops worldwide. As a sedentary root parasite, it alters the root's physiology and influences the host's phytohormonal signaling to evade defense. The sustainable management of RKN remains a challenging task. Hence, we made an attempt to investigate the nematicide activity of Bacillus velezensis VB7 to trigger the innate immune response against the infection of RKN. In vitro assay, B. velezensis VB7 inhibited the hatchability of root-knot nematode eggs and juvenile mortality of M. incognita by 87.95% and 96.66%, respectively at 96 hrs. The application of B. velezensis VB7 challenged against RKN induced MAMP-triggered immunity via the expression of transcription factors/defense genes by several folds pertaining to WRKY, LOX, PAL, MYB, and PR in comparison to those RKN-inoculated and healthy control through RT-PCR. Additionally, Cytoscape analysis of defense genes indicated the coordinated expression of various other genes linked to immune response. Thus, the current study clearly demonstrated the effectiveness of B. velezensis VB7 as a potential nematicide and inducer of immune responses against RKN infestation in tomato.

Pan-genome analysis and molecular docking unveil the biocontrol potential of Bacillus velezensis VB7 against Phytophthora infestans.

Management of late blight of potato incited by Phytophthora infestans remains a major challenge. Coevolution of pathogen with resistant strains and the rise of fungicide resistance have made it more challenging to prevent the spread of P. infestans. Here, the anti-oomycete potential of Bacillus velezensis VB7 against P. infestans through pan-genome analysis and molecular docking were explored. The Biocontrol potential of VB7 against P. infestans was assessed using a confrontational assay. The biomolecules from the inhibition zone were identified and subjected to in silico analysis against P. infestans target proteins. Nucleotide sequences for 54 B. velezensis strains from different geographical locations were used for pan-genome analysis. The confrontational assay revealed the anti-oomycetes potential of VB7 against P. infestans. Molecular docking confirmed that the penicillamine disulfide had the maximum binding energy with eight effector proteins of P. infestans. Besides, scanning electron microscopic observations of P. infestans interaction with VB7 revealed structural changes in hypha and sporangia. Pan-genome analysis between 54 strains of B. velezensis confirmed that the core genome had 2226 genes, and it has an open pan-genome. The present study confirmed the anti-oomycete potential of B. velezensis VB7 against P. infestans and paved the way to explore the genetic potential of VB7.

Deciphering the Biomolecules from Bacillus atrophaeus NMB01 Untangles the Anti-Oomycetes Action of Trioxsalen and Corynan-17-ol, Against Phytophthora infestans Inciting Late Blight of Potato.

The antagonistic Bacillus spp. is known well for the production of versatile antimicrobial biomolecules with broad spectrum of action against different types of plant pathogens. Considering the significance of metabolically active biomolecules, attempts were made to decipher the anti-oomycete nature of biomolecules produced by Bacillus atrophaeus NMB01 during di-trophic interaction with Phytophthora infestans. Ten biomolecules produced by B. atrophaeus NMB01 during di-trophic interaction with P. infestans were docked against the twelve target proteins of P. infestans. Molecular docking of biomolecules reported trioxsalen and corynan-17-ol,18,19-didehydro-10-methoxy-acetate(ester) as best hits with highest binding energy in the range of - 7.5 to - 5 kcal/mol against target proteins of P. infestans. Comparatively less binding energy was observed for commercially available fungicides mandipropamid and metalaxyl on docking against the target proteins of P. infestans. We also confirmed the direct impact of trioxsalen andcorynan-17-ol, on P. infestans under in vitro with 66% and 50% inhibition of mycelial growth of P. infestans, respectively. This is the first study attempted to untangle the role of bioactive anti-oomycete compounds produced by B. atrophaeus strain NMB01 during di-trophic interaction with P. infestans against late blight pathogen P. infestans infecting potato. From the present study, we conclude that the biomolecules, trioxsalen and corynan-17-ol, can be explored for the management of P. infestans, the incitant of late blight of potato. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01044-7.

Antifungal Volatilomes Mediated Defense Mechanism against Fusarium oxysporum f. sp. lycopersici, the Incitant of Tomato Wilt.

In this study, the volatilomes of naturally growing plant leaves were immobilized in a suitable substrate to enhance vapors' diffusion in the soil to eradicate the Fusarium wilt pathogens in Tomato. Volatilomes produced by Mentha spicata leaves immobilized in vermiculite ball was found to be effective and exhibit 92.35 percent inhibition on the mycelial growth of Fusarium oxysporum f. sp. lycopersici (FOL). Moreover, the volatilomes of M. spicata immobilized vermiculite balls were tested based on the distance traveled by the diffused volatilomes from the ball and revealed that the volatilomes of M. spicata traveled up to 20 cm distance from the center of PVC (Polyvinly chloride) chamber showed maximum reduction in colony growth of FOL at 12th day after inoculation. Tomato plants inoculated with FOL revealed increased expressions of defense gene, pathogenesis related protein (PR1) with 2.63-fold after 72 h and the gene, transcription factor (WRKY) increased with 2.5-fold after 48 h on exposure to the volatilomes of M. spicata vermiculite balls. To the best of our knowledge, this is the first report on development of volatilomes based vermiculite ball formulations. This result indicated that the volatilomes of M. spicata are promising phyto-fumigants for management of Tomato Fusarial wilt.

Differential bacterial endophytome in Foc-resistant banana cultivar displays enhanced antagonistic activity against Fusarium oxysporum f.sp. cubense (Foc).

Diverse endophytes with multiple functions exist in different banana cultivars. However, the diversity of cultivable bacterial endophytome that contributes to antifungal activity against Fusarium oxysporum f.sp. cubense (Foc) in resistant and susceptible banana cultivars is mostly unknown. In the present study, we isolated bacterial endophytes from resistant Yengambi KM5 (AAA) and susceptible banana cultivar Ney Poovan (AB) to determine the diversity of cultivable bacterial endophytes. Our study revealed the presence of 56 cultivable bacterial endophytes and 6 nectar-associated bacteria in YKM5 and 31 cultivable bacterial endophytes in Ney Poovan. The identified cultivable bacterial genera in YKM5 included Alcaligenes, Arthrobacter, Azotobacter, Acinetobacter, Agrobacterium, Bacillus, Brucella, Brevundimonas, Brachybacterium, Beijerinckia, Klebsiella, Leclercia, Lysinibacillus, Myroides, Ochrobactrum, Pseudomonas, Rhizobium, Stenotrophomonas, Serratia, and Verticiella. In Ney Poovan, the cultivable endophytic bacterial genera present were Agrobacterium, Bacillus, Bradyrhizobium, Enterobacter, Klebsiella, Lysinibacillus, Micrococcus, Ochrobactrum, Pseudomonas, Rhizobium, and Sphingobium. Thus, the composition and diversity of cultivable endophytic bacterial genera were higher in Foc-resistant YKM5. The antifungal efficacy of bacterial endophytes Brachybacterium paraconglomeratum YEBPT2 (65.5%), Brucella melitensis YEBPS3 (63.3%), Bacillus velezensis YEBBR6 (63.3%), and nectar-associated Bacillus albus YEBN2 (61.1%) from YKM5 showed the highest antifungal activity against Foc, compared with the antifungal activity of endophytes from the susceptible cultivar.

A sequel study on the occurrence of Tomato spotted wilt virus (TSWV) in cut-chrysanthemum by DAS-ELISA using recombinant nucleocapsid protein to produce polyclonal antiserum.

The tomato spotted wilt virus (TSWV) belonging to the genus Orthotospovirus, family Tospoviridae, causes severe necrotic disease in field crops and horticultural crops, resulting in considerable yield loss worldwide. The development of protein-based diagnostics is essential to track the virus transmission and prevent its spread in vegetatively propagated crops such as ornamentals. In this study, nucleocapsid (N) gene of TSWV was cloned in pET 28 a (+) expression vector. Expression of the 32 kDa recombinant TSWV-N protein was induced in BL21 (DE3) cells using 1 mM of Isopropyl ß-d-1-thiogalactopyranoside (IPTG), and was confirmed through SDS-PAGE and Western blot by fluorescent-labeled secondary antibody. The bacterial cells expressed recombinant TSWV-N protein up to a concentration of 9.48 µg/mL. The purified protein was used for immunization of a rabbit to produce specific polyclonal antiserum. The TSWV antiserum was conjugated with the enzyme alkaline phosphatase (ALP). Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA) was developed and validated against TSWV infected hosts. This antiserum specifically reacted with recombinant N protein as well as TSWV infected hosts, but not with groundnut bud necrosis orthotospovirus (GBNV) as well as capsicum chlorosis orthotospovirus (CaCV) infecting tomato and chilli plants. The coating antibody at 1 µg/mL concentration and 1:500 dilution of enzyme conjugate were found to be effective and economical in the detection of recombinant N protein of TSWV and the virus present naturally in the infected hosts. Using standardized DAS-ELISA protocol, the TSWV titer also was quantified in artificially inoculated assay hosts. Among 11 hosts tested, higher virus titer was recorded in Nicotiana tabacum (0.270 µg/100 µL), followed by Impatiens balsamiana (0.185 µg/100 µL) and Dahlia pinnata at a low virus tire of 0.083 µg/100 µL. The diagnostic reagents and protocol (DAS-ELISA) are further validated by detecting the infection of TSWV in chrysanthemum stem cuttings from six different nurseries in the hill stations of Tamil Nadu, India. The DAS-ELISA assay experimented on six varieties from four different nurseries revealed that the Mum Yellow variety had a higher percentage of TSWV infection (36 %), which was followed by the Mum White variety (33 %); both collected from Kotagiri Nursery. The same variety exhibited a higher virus titer by DAS-ELISA, an A405 value range of 0.733 (Ì´ 0.115 µg) and 0.711 (Ì´ 0.111 µg) respectively, and a total of 27 % of TSWV infection was confirmed by screening 800 stem cuttings by DAS-ELISA. The presence of TSWV was also detected in 54 (6.75 %) asymptomatic stem cuttings from different locations, and the A405 value ranged from 0.325 to 0.468. (Ì´ 0.044-0.069 µg/100 µL); this is the first reported development of immune-based diagnostics for TSWV in India. This protocol and diagnostics will be highly useful for quarantine purposes while trading large quantities of planting materials.

Role of Thrips palmi and Parthenium hysterophorus pollen in active spread of tobacco streak virus in the cotton ecosystem.

Tobacco streak virus incidence in the cotton field, cv.CO14 at Department of Cotton, Tamil Nadu Agricultural University (TNAU), Coimbatore, India was nearly 36.50 %. Cotton plants infected with TSV exhibits different types of symptoms, including necrotic spots, lesions, mosaic, purplish necrotic rings, square drying, veinal necrosis and drying of terminal shoots. The highly prevalent thrips species in this cotton ecosystem was established as Thrips palmi (60.00 %) by morphological (ESEM) and molecular methods (RT-PCR using mtCOI primers). The density of the alternate weed host, Parthenium hysterophorus, was 15.05 plants per m2 in these fields. Association of Thrips palmi with Parthenium was confirmed, when observed under environmental scanning electron microscope (ESEM), Parthenium pollen grains (i.e., average size @ 15000X =12.94 µm) were found adhering to its body. Molecular studies through RT-PCR confirmed the presence of TSV in the leaves and pollen grains of symptomatic and symptom-free Parthenium plants collected from the cotton field (cv. CO14). Therefore, the combined role of Thrips palmi and the Parthenium pollen grains in the transmission of TSV was examined; acquiring of TSV and its presence in the body of Thrips palmi instars and adults after 72 h of AAP was convincingly demonstrated using RT-PCR, NASH and qPCR. However virus acquired thrips could not transmit the virus. Pollen from TSV infected Parthenium plants when dusted on cotton (ANKUR 2110) seedlings along with virus acquired or non-acquired thrips led to symptom development 22 days after sowing. From the study it is evident that thrips only facilitate the movement of TSV borne pollen grains, and thereby contributing to active spread of the virus.