EFFECT OF DIFFERENT INOCULATION METHODS AND INOCULUM LEVELS OF MACROPHOMINA PHASEOLINA ON OKRA

Among two methods of Macrophomina phaseolina inoculation used for pathogenicity test, soil infestation method comparatively checked more plant growth of okra plants than seed infestation method. Minimum plant length and weight, as well as seed germination were observed by soil infestation method. Significantly maximum plant mortality and root infection was also occurred in soil infestation method. Seed germination, plant growth, plant mortality and root infection of okra plants were adversely affected with the increasing inoculum levels of M. phaseolina . Seed germination and plant growth were negatively correlated with inoculated pathogen population; whereas, plant mortality and root infection were positively correlated with the inoculum level of M. phaseolina .


INTRODUCTION
Among different plant pathogens attacks okra (Abelmoschus esculentus L.) Macrophomina phaseolina (Tassi) Goid. is considered as one of the most destructive soil borne pathogen of okra (Hafiz, 1986).It has very wide host range causing diseases on more than 500 cultivated and wild plant species worldwide (Jones and Canada, 1994).In Pakistan, it causes infection on more than 67 economic hosts including field crops, pulses, flowers and vegetables (Khan, 2007;Mirza and Qureshi, 1978;Shehzad et al., 1988).The fungus can also cause hallow stem, root rot, pre-emergence and post-emergence damping-off (Reuveni et al., 1983).M. phaseolina is most often seen during summer weather (Gulya et al., 2002).About 5-100% yield losses due to this disease have been reported (Vyas, 1981).M. phaseolina does not survive more than seven days in its mycelial form but it sclerotia can survive over ten months in soil (Ghaffar & Akhtar, 1968).It usually develops when soil temperature is 80-95 o F (27-35 o C) for 2 to 3 weeks (Yang and Navi, 2003).In most case, soil bone root infecting pathogens, the rate of disease development and disease severity is directly relates with the pathogen propagules present in the soils, as well as the mode of pathogen invasion (Madden, 1980;Kenerley & Bruck, I987).Keeping in view the yield losses caused by M. phaseolina in okra, the present investigation was initiated to evaluate the impact of different inoculation method and inoculum density of M. phaseolina on okra crop.

MATERIALS AND METHODS
M. phaseolina was isolated from roots and stem of diseased okra plant samples, collected from Agriculture Research Institute, Tandojam by PDA plate method.

Multiplication of inoculum:
In order to prepare large quantity of M. phaseolina inoculum, its sclerotia were obtained by growing the test pathogen on sand+wheat meal substrate.For this purpose 95 gm of sand and 5 gm of wheat meal were mixed together thoroughly followed by moistened with 10 ml sterilized water.The substrate was transferred into 250 ml conical flask and was sterilized in the autoclave at 15Lbs for 20 minutes.Left it 24 hours for cooling and 5 mm disc from actively growing M. phaseolina pure culture was added in the conical flask.Incubation was done at room temperature for 4-6 weeks with continous shaking the flask daily in order to uniform multiplication of the pathogen inoculum.After 4-6 weeks the color of the substrate in the conical flask turned black due to the sclerotial formation.The contents of the conical flash were poured onto the 15µm sieve and black tiny sclerotia present on the surface of the sieve were collected in the sterilized glass beaker for further use Fig. 1.

RESULTS
Effect of different inoculation methods: Inoculation of M. phaseolina by either method significantly affects the plant growth as well as germination and plant mortality as compared to the un-inoculated (control) plants.It was also evident from the data that soil infestation method was more aggressive for pathogenicity, as it caused significantly more reduction in plant growth as compared to the seed infestation method (Fig. 2).Soil infestation method also significantly (P<0.05)affected the germination percent and plant mortality as lowest seed germination (74.5%) and maximum plant mortality (81.19%) was recorded in plants inoculated by this method, followed by seed infestation method in which germination was 90% and plant mortality was 77.77% (Fig. 3c & 3d).Significantly (P<0.05)maximum root infection was recorded in soil infestation method (79.75%) followed by seed infestation method (69.5%) & control (3.5%) (Fig. 3e).Effect of different inoculum level: Impact of M. phaseolina on inoculated okra plants was increased with increasing inoculum density.Plant length and weight of test plants were gradually decreased with increasing inoculum level applied in the form of sclerotia (Fig. 4a &  4b).The significantly (P<0.05)minimum plant length and weight (105.16mm and 43.48 mg) was observed in plants grown in soil inoculated with test pathogen @ 50 sclerotia gram -1 soil, whereas control plants (uninoculated) showed maximum plant length and weight (121.5 mm and 57.64 mg) (Fig. 4a & 4b).Plant mortality and root infection by the M. phaseolina is positively correlated with the pathogen population and both were increased with increasing inoculum level (Fig. 4d & 4e).Significant difference (P<0.05) in terms of root infection and plant mortality was observed within different treatments (inoculum levels).The highest plant mortality (81.19%) and root infection (79.75%) was recorded in treatments where soil was inoculated with test pathogen @ 50 sclerotia gram -1 soil, followed by 40 sclerotia gram -1 soil.It was also observed that there was not much difference between inoculum level of 40 and 50 sclerotia in terms of all parameters viz., plant length, plant weight, seed germination, plant mortality and root infection (Fig. 3 &  4).DISCUSSION Pathogenicity test, carried out during present study on okra variety Sabz Pari commonly growing in Sindh province, has confirmed that Macrophomina phaseolina is an aggressive pathogen of the okra.Its inoculation on test plants significantly reduced seed germination and plant growth, and increased the plant mortality in okra.Study also revealed that among two methods evaluated for pathogenicity test, soil infestation method comparatively caused more infection as well as checked much plants growth than seed infestation method.M. phaseolina is considered as one of the devastating pathogen of the large number of crop plants, in which it caused substantial losses (Shehzad et al., 1988;Khan, 2007).Our findings were in close agreement to those reported by other research worker from elsewhere, such as Agrawal and Singh (2000) concluded that M. phaseolina was responsible for die-back and collar rot diseases, as well as pre-and post-emergence mortality in okra.Dubey and Jha (1999) and Fakir & Mridha (1985) observed that M. phaseolina caused die-back, pre-and post-emergence mortality and collar rot diseases in okra.Also El-Mohamedy (2004) reported that M. phaseolina along with Fusarium solani, and Rhizoctonia solani caused M. phaseolina damping-off and root rot diseases in okra.Similarly, Mashooda et al. (2005) also observed that M. phaseolina and Fusarium verticilloides were responsible for collar rot, seedling rot and other diseases in okra.They also observed that inoculated seed caused reduced seed germination as well as pre-and post-emergence mortality.The present study also revealed that plant growth, seed germination, plant mortality and root infection of okra plants was adversely affected with the increasing inoculum levels of M. phaseolina.Our results in confirmation to those reported Dawar and Ghaffar (1998), Moradia (2011) and McCain & Scharpf (1989) who found that increasing sclerotial population of M. phaseolina increased the infection and colonization in sunflower, groundnut and conifers, respectively.Similarly, Umamaheswari et al. (2001) observed that in groundnut root infection was severely increased by increasing inoculum density of M. phaseolina.Kenerley and Bruck (I987) also observed greatest increased in total mortality of Fraser fir seedling with increasing inoculum density of Phytophthora cinnamomi.

Fig. 1 .
Fig. 1.Scerotia of Macrophomina phaseolina.Fig. 2. Effect of Macrophomina phaseolina on growth of okra plants Effect of M. phaseolina on (a) plant length, (b) plant weight, (c) seed germination, (d) plant mortality (e) root infection of okra plants inoculated by seed infestation or soil infestation method.Means followed by different letters in respective bar are significantly different at P= 0.05.