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Nagaraja T.G. /JPR:BioMedRx: An International Journal 2013,1(7),715-717 Research Article Available online through ISSN: 2321-4988 http://jprsolutions.info

Seasonal occurrence of endomycophytes from inner bark of Putranjiva roxburghii Wall.

Nagaraja T.G. Department of Botany,The New College,Kolhapur- 416012, Maharashtra ,India.

Received on:17-03-2013; Revised on:21-04-2013; Accepted on:25-05-2013

ABSTRACT Endophytic fungi from inner bark of Putranjiva roxburghii Wall were studied in three different seasons during 2009-2010. A total of 30 endophytes were recorded during rainy season, followed by 21 each in winter and summer season. Aspergillus niger, A. flavus, Bispora punctata., Curvularia lunata., Fusarium oxysporum and Verticillium sp.,were found to be dominant endophytes followed by Drechsclera sp., Nigrospora sp., Rhizophus stolonifer and Cladosporium species.

Key-words: Endophytes, Putranjiva roxburghii, Aspergillus sp, Verticillium sp.,Bispora punctata., Drechsclera sp., Nigrospora sp., Rhizo- pus stolonifer,Fusarium oxysporum, and Cephalosporium sp,.

INTRODUCTION The term endophyte has been defined as an organism growing within auxiliary clusters with white velvety seeds, cultivated in gardens as the plant, without harming the host plant. Bacon et al., (1977) re- shade tree practically through out India. The leaves are used in the ported the close association of an endophyte (Sphacelia typhina) in treatment of colds, fever and bark is used in rheumatism. infected tall fescue (Festuca arundinacea). Similarly Latch et al., (1984) have described two seed borne endophytes in perennial ryegrass MATERIAL AND METHODS and tall fescue with Gliocladium sp., and Phialophora sp. Condia. The bark pieces of Putranjiva roxburghii Wall (Family:Putranjivaceae) . were collected from Town Hall garden, Kolhapur periodically in three As they live inside the plant tissue they utilize the nutrients and they different seasons. The bark pieces were cut at 1-2 meters above the play important role in protecting the host plant from the insects and ground level and to the depth of 1-1.5 cm in the trunk. The collected pathogens and have potential to produce novel anti microbial sec- bark samples were brought to the laboratory and surface sterilized by ondary metabolites and plants gets benefits extensively by harboring 70 percent ethanol (v/v) for 1minute followed by 1-2 minutes in 3.5 such endophytes, they promote plant growth and confer enhanced percent sodium hypochlorite solution (v/v) in a beaker, later rinsed resistant to various pathogens by producing antibiotics along with three times in distilled water for 1 minute to remove traces of sodium secondary metabolites. Endophytic organisms stimulates greater re- hypochlorite(Petrini., 1986). The outer skin was removed slowly with sistance to stress condition, alteration in physiological properties, sterilized knife and inner portion containing cortex was cut into small production of phytohormones and other compounds of biotechno- pieces of 0.2 x 0.8 mm in dimensions (Mahesh et al., 2005). Approxi- logical interests (Daniella et al., 2004). Thus endophytes advocates a mately 100 segments were cut and are plated on Nutrient Agar and good tool for the protection of host by various pathways. Therefore, PDA media mixed with septran (100 mg per litre) and incubated in a an attempt was made to screen out endophytic fungi in the bark of chamber for 21 days at 12 hours light/dark cycles at 28 +0C. The Putranjiva roxburghii Wall. petriplates are allowed to grow endophytic fungi and monitored regu- larly. Isolation was done for pure culture of the fungi from each Putranjiva roxburghii Wall, a tall evergreen tree (Family: petriplates after 18th to 20th day by sub-culturing on to appropriate Putranjivaceae) growing to the height of more than 12 meters tall media. The seasonal endomycophytic flora were identified based on with simple leaves. The fruits are ellipsoid or rounded drupes on morphological characters using standard identification manual during 2009-2010.. *Corresponding author. Nagaraja T.G. The numbers of endophytes are calculated among all petriplates. Department of Botany Percentage of colonizing frequencies were calculated according to The New College, the method prescribed by Fisher and Petrini,(1987). The dominant Kolhapur- 416012 fungi in all three seasons were estimated by the method of Kumaresan Maharashtra,India and Suryanarayan (2002).

JPR:BioMedRx: An International Journal Vol.1 Issue 7.June 2013 715-717 Nagaraja T.G. /JPR:BioMedRx: An International Journal 2013,1(7),715-717 RESULTS AND DISCUSSION Endophytes have also been shown to influence photosynthesis rates Seasonal distribution of endophytes from the inner bark of Putranjiva in host plants tall fescue plants infected by N.coenophialum, photo- roxburghii Wall.were depicted in Tables (1.2 and 3). A few studies synthesized faster and flowered earlier than uninfected plants have been carried out in endophytic mycoflora of tropical trees ( (Newman et al., 2003). Again endophyte-infected tall fescue plant Frohlich and Hyde, 1999; Nagaraja and Devakar, 2010; Nagaraja and exhibited higher survival and flowering frequency (Hill et al., 1991). Shinde, 2010). A total of 72 fungal species have been recorded in the Control of insect pests by using endophytic fungi was reported by inner bark of this tree during 2009-2010. The genera like Aspergillus Funk et al., (1983) showing protection of the perennial ryegrass Lolium sp., Bispora sp. ,Drechsclera avenaceus.,Fusarium oxysporum, perenne L against sod web worm. Mean while Wilson and Carroll Curvularia lunata., Humicola fuscoratra, Getrochium sp., Mucor (1997) investigated a systerm where endophytic fungus provokes plumbeus., Trichothecium sp., , and Verticillium sp., were dominant mortality of the gall forming insect Besbicus mirabilis. fungi during rainy season. Table: 3. Endophytic fungi isolated from inner bark of Putranjiva Table.1. Endophytic fungi isolated from inner bark of Putranjiva roxburghii Wall during summer season 2009-2010. roxburghii Wall during rainy season 2009-2010. Sr. No. Endophytes Number of Colonization Dominant Sr. No Endophytes Number of Colonization Dominant Endophytes Frequency Fungi Endophytes Frequency Fungi

1. Aspergillus niger 4 4 13.33 1. Aspergillus flavidus 4 4 19.04 2. Aspergillus flavus 4 4 13.33 2. Aspergillus japonicus 3 3 14.28 3. Bispora punctata 3 3 10 3. Aspergillus niger 3 3 14.28 4. Curvularia lunata 1 1 3.33 4. Bispora punctata 2 2 14.28 5. Cephalosporium Sp., 2 2 6.66 5. Cladosporium fluvum 2 2 9.52 6. Diplococcium Sp., 2 2 6.66 6. Curvularia lunata 3 3 14.28 7 Fusarium oxysporum 2 2 6.66 7. Fusarium oxysporum 1 1 4.76 8. Geotrichum Sp., 1 1 3.33 8. Memnoniella Sp., 1 1 4.76 9. Humicola fuscoatra 1 1 3.33 9. Verticillium Albo-atrum 2 2 9.52 10. Mucor plumbeus 2 2 6.66 Total isolation 21 21 11. Rhizopus stolonifera 1 1 3.33 12. Verticillium Sp., 3 3 10 Total segments: 100 13. Verticicladiella Sp., 2 2 6.66 Total endophytes: 21 14 Trichothecium Sp., 2 2 6.66 Total isolates 30 30 Endophytic fungi associated with grass have been shown to protect Total segments; 100 grasses against pests and diseases (Clay, 1989). The endophytic Total endophytes; 30 fungi like Fusarium sp and Trichoderma sp.,are basically pathogenic A total of 21 species of hypomyceteous fungi were recorded in the to crop, but sometimes they get modified by mutation and grow into inner bark of Putranjiva roxburghii Wall. During winter seasion non-pathogenic endophytes (Freeman and Rodriquez, 1993). Mean- from Nov 2009 to Jan 2010 (Table. 2). Aspergillus niger, A.japonicus., while some root colonizing plant beneficial fungi such as Fusarium Cladosporium sp., Nigrospora sphaeia., Paecilomyces sp.,and Ver- sp., and Trichoderma sp which have developed symbiotic relation- ticillium albo-atrum were dominant fungi followed by Fusarium ship with host plant (Haas and Defego, 2005).So our results coin- oxysporum.;, F. moniliformis.,Choanosporium sp.and sterile myce- cides with their findings. lia. Mean while a few endophytes were recorded in summer period from Feb 2010 - May 2010 (Table. 3 ). Aspergillus sp., and Bispora The toxic products synthesized by endophytes in woody plants and punctata, , and Cladosporum sp.,were dominant followed by that were able to modify growth and death rates in larvae of the Memnoniella sp., spruce bud worm C. fumiferanna feeding on balsam fir (Calhoun et al., 1992). The endophytes were identified as Phyllosticta sp.,and Table: 2. Endophytic fungi isolated from inner bark ofPutranjiva Hormonema dematioides and the toxic compounds were mainly roxburghii Wall during winter season 2009-2010. heptelidic acid and regulosine, even tremorgenic toxin in tropical Sr. no Endophytes Number of Colonization Dominant woody plant infected with an endophytic fungus from the Phomopsis Endophytes Frequency Fungi (Billis, et al., 1992) recorded. Antibiotic phomol was isolated from 1. Aspergillus flavus 3 3 14.28 fermentation by Phomopsis sp., endophytic fungus from Erythrina 2. Aspergillus japonicus 4 4 19.14 crista-galli (Webber, 1981). Thus endophytes providing protection 3. Aspergillus niger 4 4 19.14 against pathogens as well as they are potential biocontrol agents and 4. Cladsporium fulvum 1 1 4.76 5. Choanephora Sp., 2 2 9.52 could be utilized to protect tissue culture plants before they are trans- 6. Fusarium oxysporum 2 2 9.52 planted to the field. 7. Fusarium moniliformis 2 2 9.52 8. Nigrospora sphaeria 1 1 4.76 9. Paeilomyces Sp., 1 1 4.76 ACKNOWLEDGEMENT 10. Verticillium Albo-atrum 1 1 4.76 The author is very much thankful to Co-ordinator, Department of Total isolates 21 21 Agro Chemicals and Pest Management, Shivaji University, Kolhapur Total segments; 100 for providing laboratory facilities. Total endophytes; 21

JPR:BioMedRx: An International Journal Vol.1 Issue 7.June 2013 715-717 Nagaraja T.G. /JPR:BioMedRx: An International Journal 2013,1(7),715-717

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JPR:BioMedRx: An International Journal Vol.1 Issue 7.June 2013 715-717