Ramseena, S R
Endospore-forming endophytic bacteria for the management of tospovirus infection in COWPEA(vigna unguiculata ssp. sesquipedalis) - Vellayani Department of plant pathology, college of agriculture 2023 - 118p.
MSc
The study entitled “Endospore-forming endophytic bacteria for the management of
Tospovirus infection in cowpea (Vigna unguiculata ssp. sesquipedalis)” was carried out at
College of Agriculture, Vellayani during 2021-2023 with an objective of evaluating the
efficacy of Endospore-forming Endophytic Bacteria (EEB) for the management of Tospovirus
causing bud necrosis in cowpea (Vigna unguiculata ssp. sesquipedalis).
The virus was sap transmissible and was maintained in local lesion host,
Chenopodium amaranticolor and susceptible cowpea variety, Pusa Komal by mechanical
inoculation using potassium phosphate buffer. On mechanical inoculation to chenopodium
plants, cowpea Tospovirus isolate took 4-5 days for symptom development and produced
chlorotic and necrotic lesions. In cowpea variety Pusa Komal it took 8 days for symptom
development and produced both local and systemic infections. The virus that causes cowpea
bud necrosis disease was identified serologically using ELISA and DIBA, and it was
discovered that Tospovirus isolation from several cowpea samples demonstrated a close
relationship with Watermelon silver mottle virus (WSMoV). Molecular detection was done by
RT-PCR and an expected amplicon of size 477–500 bp was obtained using the primer specific
to the coat protein gene of Groundnut bud necrosis virus (GBNV). Serological and molecular
detection confirmed that the virus associated with bud necrosis of cowpea belong to the
serogroup IV which is Watermelon silver mottle orthotospovirus.
Screening of five different Bacillus strains was done for evaluating its efficacy in
managing cowpea bud necrosis disease. Bacillus pumilus VLY17, Bacillus amyloliquefaciens
VLY24, Bacillus velezensis PCSE10, Bacillus amyloliquefaciens CBSE5 and Bacillus
velezensis CBRE5 were used for screening. Pre and post inoculation with EEBs in C.
amaranticolor and susceptible cowpea variety Pusa Komal were carried out and B. pumilus
VLY17 treated plants demonstrated reduced number of local lesions (4) in C. amaranticolor,
when compared to that of virus inoculated control with a significantly higher number of local
lesions (11). In cowpea, the plants pre colonised with B. pumilus VLY17 were observed with
low vulnerability index of (V I) (20) compared to untreated plants with a V I of 66.6. Hence,
B. pumilus VLY17, with good disease-suppressing ability, was selected and further evaluated
against the virus in tolerant cowpea variety, Githika, to standardise the effective method of
application. The most efficient strategy of treating EEB, according to the analysis of different
application techniques, was seed priming followed by foliar spray and drenching with a
suspension of B. pumilus at 108 CFU per ml at cotyledonary leaf stage of cowpea plants
130
compared to the untreated ones. The number of branches (19), fresh weight (163.6 g) and
number of pods (19) obtained from B. pumilus VLY17 treated plants showed a significant
increase than that of the virus control plants. The treated plants also flowered 3-4 days earlier
than the virus control and exhibited considerable resistance towards further pest and disease
occurrence.
When analysed the enzyme activity over 3 weeks in different treatments, enzyme
activity of polyphenol oxidase and phenylalanine ammonia-lyase was found to be higher at
second week and peroxidase activity was higher at the third week in treated plants. On
analysis of the average of enzyme activity over 3 weeks, peroxidase (74.1 µg/g of leaf tissue
min -1
), polyphenol oxidase (6.23 µg/g of leaf tissue min -1
) and phenylalanine ammonialyase (72.7 µg of cinnamic acid per gram of leaf tissue) were found to be high in treatment,
where seed priming followed by soil drenching and foliar spray at cotyledonary leaf stage
prior to the virus inoculation was applied. Total protein content (49.9 µg of BSA per gram of
leaf tissue) was also found to be higher in treated ones when compared to the untreated ones
at second week. Expression of defense genes such as NPR1, Coi1, PAL and BGL were
analysed in treated and virus control plants by RT-PCR and ImageJ software. The analysis
showed that seed treatment followed by foliar spray and drenching with B. pumilus VLY17 at
the cotyledonary leaf stage of cowpea plants prior to virus inoculation, gave the highest
expression of defense genes such as NPR1 (46.18), Coi1 (31.56) and PAL (25.59) in terms of
band peak percentage compared to the virus inoculated and absolute control plants. The BGL
gene expression (33.55) was found to be less in B. pumilus VLY17 seed primed plants than
the virus control plants. There are reports establishing that BGL gene expression is controlled
by the virus in infected plants as it helps in the movement of the virus by hydrolysis of
callose, deposited in plasmodesmata, formed as a part of defense due to virus infection.
Hence, less activity of BGL gene is indicating more defense as it can hinder the movement of
virus.
The research highlights the biocontrol potential of B. pumilus VLY17 to reduce the
disease severity by enhancing the expression of defense genes and by promoting growth and
yield parameters of cowpea plants. Seed priming with B. pumilus VLY17 for 4 hours
followed by soil drenching and foliar spray at cotyledonary stage of cowpea is the best
method of application of B. pumilus VLY17 for the management of bud necrosis disease in
cowpea
English and malayalam
plant pathology
Endospore-forming endophytic bacteria
632.3 / RAM/EN PG
Endospore-forming endophytic bacteria for the management of tospovirus infection in COWPEA(vigna unguiculata ssp. sesquipedalis) - Vellayani Department of plant pathology, college of agriculture 2023 - 118p.
MSc
The study entitled “Endospore-forming endophytic bacteria for the management of
Tospovirus infection in cowpea (Vigna unguiculata ssp. sesquipedalis)” was carried out at
College of Agriculture, Vellayani during 2021-2023 with an objective of evaluating the
efficacy of Endospore-forming Endophytic Bacteria (EEB) for the management of Tospovirus
causing bud necrosis in cowpea (Vigna unguiculata ssp. sesquipedalis).
The virus was sap transmissible and was maintained in local lesion host,
Chenopodium amaranticolor and susceptible cowpea variety, Pusa Komal by mechanical
inoculation using potassium phosphate buffer. On mechanical inoculation to chenopodium
plants, cowpea Tospovirus isolate took 4-5 days for symptom development and produced
chlorotic and necrotic lesions. In cowpea variety Pusa Komal it took 8 days for symptom
development and produced both local and systemic infections. The virus that causes cowpea
bud necrosis disease was identified serologically using ELISA and DIBA, and it was
discovered that Tospovirus isolation from several cowpea samples demonstrated a close
relationship with Watermelon silver mottle virus (WSMoV). Molecular detection was done by
RT-PCR and an expected amplicon of size 477–500 bp was obtained using the primer specific
to the coat protein gene of Groundnut bud necrosis virus (GBNV). Serological and molecular
detection confirmed that the virus associated with bud necrosis of cowpea belong to the
serogroup IV which is Watermelon silver mottle orthotospovirus.
Screening of five different Bacillus strains was done for evaluating its efficacy in
managing cowpea bud necrosis disease. Bacillus pumilus VLY17, Bacillus amyloliquefaciens
VLY24, Bacillus velezensis PCSE10, Bacillus amyloliquefaciens CBSE5 and Bacillus
velezensis CBRE5 were used for screening. Pre and post inoculation with EEBs in C.
amaranticolor and susceptible cowpea variety Pusa Komal were carried out and B. pumilus
VLY17 treated plants demonstrated reduced number of local lesions (4) in C. amaranticolor,
when compared to that of virus inoculated control with a significantly higher number of local
lesions (11). In cowpea, the plants pre colonised with B. pumilus VLY17 were observed with
low vulnerability index of (V I) (20) compared to untreated plants with a V I of 66.6. Hence,
B. pumilus VLY17, with good disease-suppressing ability, was selected and further evaluated
against the virus in tolerant cowpea variety, Githika, to standardise the effective method of
application. The most efficient strategy of treating EEB, according to the analysis of different
application techniques, was seed priming followed by foliar spray and drenching with a
suspension of B. pumilus at 108 CFU per ml at cotyledonary leaf stage of cowpea plants
130
compared to the untreated ones. The number of branches (19), fresh weight (163.6 g) and
number of pods (19) obtained from B. pumilus VLY17 treated plants showed a significant
increase than that of the virus control plants. The treated plants also flowered 3-4 days earlier
than the virus control and exhibited considerable resistance towards further pest and disease
occurrence.
When analysed the enzyme activity over 3 weeks in different treatments, enzyme
activity of polyphenol oxidase and phenylalanine ammonia-lyase was found to be higher at
second week and peroxidase activity was higher at the third week in treated plants. On
analysis of the average of enzyme activity over 3 weeks, peroxidase (74.1 µg/g of leaf tissue
min -1
), polyphenol oxidase (6.23 µg/g of leaf tissue min -1
) and phenylalanine ammonialyase (72.7 µg of cinnamic acid per gram of leaf tissue) were found to be high in treatment,
where seed priming followed by soil drenching and foliar spray at cotyledonary leaf stage
prior to the virus inoculation was applied. Total protein content (49.9 µg of BSA per gram of
leaf tissue) was also found to be higher in treated ones when compared to the untreated ones
at second week. Expression of defense genes such as NPR1, Coi1, PAL and BGL were
analysed in treated and virus control plants by RT-PCR and ImageJ software. The analysis
showed that seed treatment followed by foliar spray and drenching with B. pumilus VLY17 at
the cotyledonary leaf stage of cowpea plants prior to virus inoculation, gave the highest
expression of defense genes such as NPR1 (46.18), Coi1 (31.56) and PAL (25.59) in terms of
band peak percentage compared to the virus inoculated and absolute control plants. The BGL
gene expression (33.55) was found to be less in B. pumilus VLY17 seed primed plants than
the virus control plants. There are reports establishing that BGL gene expression is controlled
by the virus in infected plants as it helps in the movement of the virus by hydrolysis of
callose, deposited in plasmodesmata, formed as a part of defense due to virus infection.
Hence, less activity of BGL gene is indicating more defense as it can hinder the movement of
virus.
The research highlights the biocontrol potential of B. pumilus VLY17 to reduce the
disease severity by enhancing the expression of defense genes and by promoting growth and
yield parameters of cowpea plants. Seed priming with B. pumilus VLY17 for 4 hours
followed by soil drenching and foliar spray at cotyledonary stage of cowpea is the best
method of application of B. pumilus VLY17 for the management of bud necrosis disease in
cowpea
English and malayalam
plant pathology
Endospore-forming endophytic bacteria
632.3 / RAM/EN PG