S. Shankara Bhat

Study of die back disease incidence of neem in Karnataka, India and PCR based identification of the isolates

A disease survey of die back of neem was done in different agroclimatic regions of Karnataka, India using Global Positioning System (GARMIN 12). Twigs of Azadirachta indica (Neem) infected with die back were collected from different regions of Karnataka, India and they were further analysed to determine the pathogen. Phomopsis azadirachtae the causal organism was isolated on malt extract agar from die back infected neem twigs. They were identified by conventional and molecular methods. Phomopsis genus specific primers (5.8S r-DNA) were then used for the detection of P. azadirachtae, the causative agent of die back of neem by polymerase chain reaction (PCR). Studies revealed the amplification of expected 141 bp DNA in P. azadirachtae isolated from the diseased trees of different regions of Karnataka indicating the causal organism of die back disease on neem. Studies revealed a very high incidence of die back in most of the places of Karnataka. This is the first report on disease incidence of die back of neem. Hand held GPS was used in the study which helps in continuous monitoring of the diseased trees.

In vitro screening of systemic fungicides against Phomopsis azadirachtae, the incitant of die-back of neem

Abstract Phomopsis azadirachtae Sateesh, Bhat & Devaki is the causal organism of die-back of neem (Azadirachta indica A.Juss.) which is, presently, a major crippling disease of neem in India. Six systemic fungicides such as Bavistin (carbendazim), Contaf (hexaconazole), Beam (tricyclazole), Fuji-one (isoprothiolane) Roko (thiophanate methyl) and Downymil (metalaxyl) were evaluated against P. azadirachtae under in vitro conditions. Colony diameter, mycelial dry weight, pycnidial formation and the germ tube length of the pathogen were the parameters studied. The results indicated that carbendazim was the most effective in inhibiting the growth followed by thiophanate methyl. Among the different concentrations tested, carbendazim at 0.25 ppm and thiophanate methyl at 0.75 ppm were optimum for controlling the growth of the pathogen. Both these fungicides can be utilized for the control of die-back of neem.

Growth of Sclerospora graminicola in host tissue cultures

Callus from shoot tips of Pennisetum typhoides (bajra) infected with Sclerospora graminicola was grown on a modified Whites medium. Non-infected callus cultures obtained from seed and stem tips could be infected with aseptic sporangia, but not with oospores. Dense, felt-like aerial mycelium was produced on infected callus and frequently spread onto the medium, but it did not grow independently. Invasion of newly formed callus by mycelium of S. graminicola was generally rapid. Mycelium was normally coenocytic, but occasionally septate. Both asexual and sexual spores, typical of the species, were formed in callus tissue. Oogonia formed on the aerial mycelium did not develop into oospores; only oospores on submerged hyphae matured. The mycelium retained its infectivity through 5 years of subculturing. Seedlings of P. typhoides and non-infected callus cultures of P. pedicellatum, Eleusine coracana, and Dactyloctenium aegyptium were successfully infected with mycelium from infected callus cultures of P. typhoides.

PCR-based detection of Phomopsis azadirachtae in die-back affected neem seeds.

Abstract Phomopsis azadirachtae Sateesh, Bhat & Devaki is the incitant of die-back disease of neem trees. Delayed appearance of conidia and presence of other microorganisms in the neem tissues are the obstacles in the rapid and accurate identification of P. azadirachtae. This work was carried out to develop a methodology for rapid detection of the pathogen in diseased tissues especially in the neem seeds. rDNA sequences of many Phomopsis spp. were retrieved from the database and were subjected for multiple alignment to select a 179 bp conserved sequence. This was used to design Phomopsis specific primer pair (Forward and Reverse) having the potential to produce a 154 bp product in PCR. The primer pair was utilised to detect the presence of P. azadirachtae in diseased neem seeds and other tissues. This is the first report on the PCR-based detection of P. azadirachtae directly in die-back diseased neem tissues. This method can be employed for rapid and reliable detection of P. azadirachtae in die-back affected neem seeds. Hence it will have very good application in seed health testing laboratories.

An efficient method for culturing downy mildew fungi on host callus

A method of obtaining conidia of Peronosclerospora sorghi and transferring them to host callus to obtain contaminant-free dual culture is described.

Detection of Phomopsis azadirachtae from dieback affected neem twigs, seeds, embryo by polymerase chain reaction

Abstract Twigs and seeds of Azadirachta indica (Neem) infected with dieback disease, collected from different regions of Karnataka, India were analysed to determine the pathogens. Phomopsis azadirachtae, the causal agent of dieback disease of neem was found to be seed borne. Phomopsis azadirachtae was isolated on PDA and MEA from dieback-infected neem twigs, seeds and embryo. Phomopsis genus-specific primers (5.8S r-DNA) were then used for the detection of Phomopsis azadirachtae, the causative agent of dieback of neem by polymerase chain reaction (PCR). Reactions were performed on DNA isolated from twigs, seeds and embryo of dieback-affected neem trees. Studies revealed the amplification of expected 141 bp DNA in Phomopsis azadirachtae isolated from various parts of diseased trees indicating the causal organism of dieback disease on neem. Isolation and identification by conventional technique takes around 15 – 21 days, whereas the present technique is capable of detecting very low propogules within 4 – 5 days.

Intraspecific variability in Phomopsis azadirachtae infecting neem.

Abstract Phomopsis azadirachtae Sateesh, Bhat and Devaki is the causal agent of die-back disease of neem. Six isolates of P. azadirachtae collected from different geographical regions of Tamilnadu were subjected to SDS-PAGE to study the variation among the isolates. Mycelial soluble proteins extracted from the six isolates exhibited marked variations in their electrophoretic protein profile. A few bands were common to all the isolates and each isolate also had a few specific bands. Soluble proteins were resolved into 42 bands of different molecular weights. Similarity index obtained ranged from 29.63% to 59.26%. Above findings indicate the existence of variability in P. azadirachtae isolates and its heterogeneous nature, revealing the genetic diversity of the pathogen.

A survey of die-back disease of neem in Tamil Nadu, India and PCR-based confirmation of the isolates

A survey of die-back disease of neem was done in different agro climatic regions of Tamil Nadu, India using Global Positioning System (GARMIN 12). Twigs of Azadirachta indica (Neem) infected with die-back were collected from different regions of Tamil Nadu, India and they were further analyzed to determine the pathogen. Phomopsis azadirachtae the causal organism was isolated on malt extract agar from die-back infected neem twigs. They were identified by conventional and molecular methods. Phomopsis genus specific primers (5.8S r-DNA) were then used for the confirmation of P. azadirachtae – the causative agent of die-back of neem by Polymerase chain reaction (PCR). Studies revealed the amplification of expected 141bp DNA in P. azadirachtae isolated from the diseased trees of different regions of Tamil Nadu confirming the causal organism of die-back of neem. Studies revealed a very high incidence of die-back in most of the places of Tamil Nadu. Hand held GPS was used in the study which would help in continuous monitoring of the diseased trees.

Germination of oospores of Peronosclerospora sorghi

An improved and reliable method of germinating oospores of Peronosclerospora sorghi is described. Germination of oospores occurred in response to root exudates of susceptible and resistant host as well as non-host plants. A germination-triggering factor was present in the root exudates of all the plants tested. The root exudate was found to be as effective as furfural.

In vitro efficacy of plant essential oils against Phomopsis azadirachtae – the causative agent of die-back disease of neem

Seven essential oils – Rosemary, Wintergreen, Teatree, Patchouli, Palmarosa, Geranium and Eucalyptus were used to determine the in vitro antifungal activity on Phomopsis azadirachtae. This organism is responsible for the destructive die-back disease of neem. The oils mentioned above were screened using the food poisoning technique. All the seven oils showed considerable antifungal activity against P. azadirachtae. After the conduction of the tests using the oils, the results were such that Wintergreen, Geranium and Palmarosa showed complete or 100% inhibition of mycelial growth at a concentration of 2500 ppm. Among the oils tried, Palmarosa proved to be most potent showing complete inhibition of mycelia at 500 ppm. This really suggests that plant sources possess antifungal activity and can be effectively used to treat and manage plant diseases thereby circumventing the fungal spread in our environment.