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.