K. Girish

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.

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.

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.

Synergistic effect of combinations of fungicides and bacterial extracts against Phomopsis azadirachtae causing die-back of neem

Phomopsis azadirachtae causes die-back of neem and this disease is presently a major devastating disease of neem in India, resulting in the reduction of life expectancy and flower production. Development of effective, eco-friendly management strategies against this disease is most important. Two systemic fungicides carbendazim and thiophanate- methyl were combined with ethyl acetate extract of antagonistic bacteria Pseudomonas aeruginosa culture filtrate at different concentrations viz., 100F: 0E, 80F: 20E, 60F: 40E, 50F: 50E, 40F: 60E, 20F: 80E, 0F: 100E and evaluated against P. azadirachtae under in vitro conditions. The parameters studied were colony diameter, mycelial dry weight, pycnidial formation and the germ tube growth of the pathogen. The effect of these combinations on neem seed germination and seed-borne pathogen was also tested. The results indicated that the combinations tested were effective in inhibiting the growth of the pathogen in vitro. The combinations also inhibited the growth of P. azadirachtae from die-back infected neem seeds and had no significant negative effect on neem seed germination. These combinations could be utilized for the integrated control of die-back of neem.