M. NAGALAKSHMI DEVAMMA, S. THAHIR BASHA AND N.P. ESWARA REDDY*
Department of Plant Molecular Biology and Biotechnology, S.V. Agricultural College, Acharya NG Ranga Agricultural University, Tirupati-517502, India
Mango is the most important fruit covering about 35 per cent of area and accounting of 22 per cent total production of total fruits in India, which is highest in the world with India’s share of about 49.62 per cent, is severely affected by mango anthracnose caused by Colletotrichum gloeosporioides a devastating disease responsible for 30 – 60 per cent post harvest losses. Out of 276 (bacteria – 247 + fungi – 29) leaf endophytes screened against the aggressive pathogen, the potential endophytic bacteria were cloned and identified. Further, the talc based formulations of novel bacteria were developed to control the anthracnose disease and also to minimize the risk in use of fungicides. As a part of common practice, the formulations/fungicides are applied prior to the infection and during the favourable environmental conditions, the fungus which is in dormant state becomes active and cause disease symptoms. As the environmental conditions and geographical factors plays a major role, it is necessary to understand the favourable temperature for the growth of the fungus in order to recommend the scheduled spray time to the farmers. In the present study, an investigation into the significance of temperature with respect to pathogen growth (sporulation) was studied using Analysis of Variance (ANOVA) with Post hoc test using advanced statistical software SAS Ver. 9.2. The research findings concluded that the temperature between 25°C – 3 0°C was most favourable for the growth of the C. gloeosporioides and also revealed that there is a significant difference between three temperature conditions of radial growth, growth rate and sporulation (p < 0.0001). These findings help in allowing the farmers to take suitable measures in order to control the anthracnose disease and fetch more income.
Mango; anthracnose disease; temperature; SAS.
Mango (Mangifera indica L.) is native to India and South East Asia. India is the largest producers of mangoes in the world when compared to half of the global production and the largest exporter. Andhra Pradesh is a leading state in production and productivity of mango in India. Devastating disease like anthracnose caused by Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. reduce quality of fruit produced under wet or very humid conditions. The post harvest phase is the most economically significant throughout the world. Post harvest produce and responsible for 30 to 60 per cent of harvest losses. It is the major pre and postharvest disease of mango (Arauz, 2000), and can result in serious decay of fruit during marketing and after sale. The incidence of this reach almost 100 per cent in fruits produced under wet or very humid conditions. The post harvest phase is the most economically significant throughout the world.
Post harvest thermal and chemical treatments reduces anthracnose severity of the fruits but the adverse effect of synthetic chemical residues on human health, environment and the development of resistance in the pathogen to chemicals used for controlling the disease have lead to intensified efforts to develop alternative methods. Biological control using microbial antagonists has emerged as one of the most promising alternatives, used either alone or as integrated control strategy to reduce the use of fungicides. But the information on the growth of the fungal pathogen with respect to the temperatures and geographical regions is scanty which directly linked with the spray schedules. Now, to identify any variations in the growth parameters of the pathogen at different temperatures provides much insight information in controlling the disease is the need of the hour. The meteorological factors which form the basis of such predictions usually are prevailing temperatures and wet periods. Although it is known that temperature affects spore germination and infection, which in turn influence disease development, the period required for infection will be largely influenced by prevailing temperatures, provided wetting is unlimited. If the wetting period is short, infection will only be successful if the temperature is optimal. Considering the severity of the disease and the losses associated with it, an investigation was made to study the growth parameters of C. gloeosporioides may assist in better understating the relationship of the growth and temperature in specific geographical locations for better timing of fungicide applications.
Mango leaves infected with Colletotrichum gloeosporioides Penz., were collected during the roving survey was conducted to estimate the disease incidence in major mango growing regions of Andhra Pradesh for the estimation of per cent disease incidence. Isolations were made from the leaves showing typical symptoms of anthracnose disease by tissue segment method (Rangaswami and Mahadevan, 1999) on potato dextrose agar medium (PDA). These bits were transferred to sterile discs of blotting paper. The dried bits were subsequently transferred to sterile PDA medium in Petri plates under aseptic conditions. The Petri plates were incubated at 28 ± 2°C and the fungal growth was observed after seven days. Purification and identification of pathogen
The fungal colonies developed were purified by single spore isolation method (Rangaswami and Mahadevan, 1999) and the pathogen was identified based on its mycelia and conidial characteristics as per standard mycological keys (Barnett and Hunter, 1972).
Potato dextrose agar medium (PDA) was used to assess the differential growth among the isolates of the fungus. Morphological characteristics such as colour of the colony and size of the conidia (length and width) were recorded for each isolate. Length and width of the conidia were measured by Motic image software programme inbuilt in Motic BA200 binocular microscope.
Measurement of radial growth of the fungus
The variation among the isolates of C. gloeosporioides in colony growth, the radial growth of the fungus in each Petri plate (replications) was recorded
after seven days of incubation i.e., when the growth of the fungus in some of the Petri plates was maximum. The colony growth was measured along two diameters at right angles and averaged.
The radial growth of the fungus in each Petri plate (replications) for each isolate was measured at every 24 hours interval in order to determine the growth rate.
Sporulation of the individual isolates of fungus on PDA medium was assessed. Five discs of each 5 mm diameter size were cut with a sterile cork borer at a radius of 20 mm from the centre of the growth. The discs were placed in a test tube containing 10 ml of sterile distilled water and macerated with the help of glass rod so as to get uniform dispersion of conidia. From this spore suspension, a drop was transferred on to the counting chamber of the haemocytometer and average number of spores per ml was determined. The conidial population was categorized as poor, average, good and excellent as given below:
Among three different temperatures tested, the temperature 26°C – 30°C was found to be most favorable temperature for the growth of fungal pathogen (radial growth, growth rate and sporulation). The effect of temperature on the growth parameters of fungal pathogen C. gloeosporioides collected from all three different regions of Andhra Pradesh showed significant difference with respect to different temperatures (p<0.0001) (Table 1 and Fig 1). Among all the three different regions of Andhra Pradesh, the fungal parameters ware found to be more or less similar. Hence, the results conclude that the growth parameters of C. gloeosporioides showed that there is no significant difference with respect to different geographical locations (p>0.05) (Table 2 and Fig. 2). The fungal growth parameters viz., radial growth, growth rate
and sporulation were found to be higher at 26°C – 30°C compared with 20°C – 25°C and 31°C – 35°C in all three regions has shown that significance difference (p< 0.0001) (Table 3 and Fig. 3).
In Chittoor region, the radial growth has higher mean (87.18) at 26°C – 30°C when compared with the mean values of 20°C – 25°C (61.82) and 31°C – 35°C (53.63). The growth rate has higher mean (12.45) at 26°C–30°C when compared with the mean values of 20°C – 25°C (9.78) and 31°C – 35°C (7.32). The sporulation has higher mean (5.29) at 26°C – 30°C when compared with the mean values of 20°C – 25°C (2.60) and 31°C – 35°C (1.47). The results stated that there is a significant effect of temperature on growth parameters of fungal pathogen C. gloeosporioides in three different regions of Andhra Pradesh (p<0.0001).
In Kadapa region, the radial growth has higher mean (85.67) at 26°C – 30°C when compared with the mean values of 20°C – 25°C (64.20) and 31°C – 35°C (52.53). The growth rate has higher mean (12.91) at 26°C – 30°C when compared with the mean values of 20°C – 25°C (9.57) and 31°C – 35°C (7.33). The sporulation has higher mean (4.92) at 26°C – 30°C when compared with the mean values of 20°C – 25°C (2.30) and 31°C – 35°C (1.76). The results stated that there is a significant effect of temperature on growth parameters of fungal pathogen C. gloeosporioides in three different regions of Andhra Pradesh (p<0.0001).
In Kurnool region, the radial growth has higher mean (86.52) at 26°C – 30°C when compared with the mean values of 20°C – 25°C (67.72) and 31°C – 35°C (55.38). The growth rate has higher mean (13.14) at 26°C – 30°C when compared with the mean values of 20°C – 25°C (10.93) and 31°C – 35°C (9.54). The sporulation has
higher mean (5.06) at 26°C – 30°C when compared with the mean values of 20°C – 25°C (2.84) and 31°C – 35°C (1. 74). The results stated that there is a significant effect of temperature on growth parameters of fungal pathogen C. gloeosporioides in three different regions of Andhra Pradesh (p<0.0001).
The current study findings are in accordance with earlier reports by Banik et al., (1988) and Quesada and Lopez (1980) observed that 28°C favoured the good growth of C. gloeosporioides, whereas Rajak (1983) and Ekbote (1994) recorded maximum growth of C. gloeosporioides at 25°C and 29°C respectively. Quimio and Quimio (1975) and Ahmed (1985) who recorded good growth of C. gloeosporioides at a temperature range of 20 – 30°C. The present investigations prove that C. gloeosporioides shall sporulate between a temperature range of 25°C – 28°C. Similar reports on good sporulation of C. gloeosporioides at a temperature range of 15°C – 35°C, optimum being between 20°C – 30°C recorded by Ahmed (1985) whereas 28°C was best temperature as per findings made by Quesada and Lopez (1980).
The research findings concluded that the optimal temperature for germination was between 25°C and 30°C in the all three regions of Andhra Pradesh from where the C.gloeosporioides isolates were collected. Poor germination was recorded at both the 20°C – 25°C and 31°C–35°C temperatures and the study revealed in understanding the sensitivity of temperature for the mycelial growth and conidial germination of the fungal pathogen. This information helps the researchers as well as farmers for the prediction of pathogen infection during the season and would encourage in planning spray schedules.