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Dutta et al., Vegetos 2017, 30:2 DOI: 10.4172/2229-4473.1000236
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Research Article
Effect of Climatic Parameters on Persistence of Metarhizium anisopliae Pranab Dutta1*, Himadri Kaushik1**, H. Singh2, R P Bhuyan3, DJ Nath4 and KC Puzari1
Abstract The work has been done on the study related to the persistence of a potential entomopathogen Metarhizium anisopliae outside of their insect host as there is little or no knowledge about the biology. To resolve this question, the study was conducted in organic and inorganic tea ecosystem of Assam. Results have shown that the entomopathogen remained viable and detectable for 1 year and a month in both soil and air in inorganic tea ecosystem. While it was detectable for 1 year and a month in soil and for approximately 12 months in air in organic tea ecosystem, with negative periods between. M. anisopliae this did not persist as epiphyte in the vegetative cover of both the tea ecosystem even for a month after its application. However, its persistence is highly depended upon all the environmental parameters namely temperature, rainfall, humidity, BSSH, evaporation and wind speed. Moreover, soil biological and physico-chemical properties like soil pH, available NPK, exchangeable cations, soil organic carbon and microbial biomass carbon were found to be increased after its application in the studied area. The results of this study showed that the population of M. anisopliae has the potential of accumulating in the soil and air under proper environmental conditions. Conidia present in these areas decay after one year so, fungal spraying must be done accordingly to achieve a better control. We could not able to detect positive colonies of M. anisopliae as an epiphyte in the vegetative cover of the studied area. If conidia are applied on the abaxial surface of leaves, persistence of the entomopathogen could be enhanced on phylloplanes. Keywords Ecosystem; Metarhizium; Tea; Persistence; Phylloplanes; Viability
Introduction Tea, Camellia sinesis (L.) O. Kuntze has been domesticated and grown for nearly two centuries, but the place of origin of tea plant is still a matter of speculation. Globally tea is growing in an area of 5 mha with a production of 3.527 MT. India is one of the largest producer and consumer of tea with an annual production of 967.6 million kg from a production area of 540722.346 ha. India consumes 786 mkg of tea per
*Corresponding authors: Pranab Dutta, Department of Plant Pathology, Assam Agricultural University, Jorhat-785013, Assam, India , E-mail:
[email protected] Himadri Himadri Kaushik, Department of Plant Pathology, Assam Agricultural University, Jorhat-785013, Assam, India; E-mail:
[email protected] Received: March 13, 2017 Accepted: April 11, 2017 Published: April 14, 2017
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Vegetos- An International Journal of Plant Research a SciTechnol journal
year. In India, the major part of the produce is concentrating towards the North-Eastern part including Assam, Manipur, Meghalaya, Mizoram, Tripura, Nagaland and Arunachal Pradesh, which are the traditional areas for tea plantation and considered to be the best place for tea cultivation. Assam is the only region with native tea plants in India and second in the world after southern region of China. Assam is well known for its black tea produce from large leaves of tea plants. The great Brahmaputra River side of this region is considered to be the world’s largest tea-growing area of 312,210 hectare with a production of 507 mkg. The favourable tropical climate with a high downfall during the monsoon of Assam gives a unique feature to its tea, a malty taste for which this tea is well known in the world. Recent publication showed that there are 77 numbers of organic tea garden in Assam which have increased by 24 numbers over 2007 with a percentage growth of 45.25%. Similarly, in 2007 the total area under organic tea was 10,208 ha and it has reached to 15,726 ha in 2013 with percentage growth of 54.05% along with total production of 11.09 mkg with a percentage growth increase of 45.15 over 2007 (7.64 mkg) [1]. Being a perennial crop it is attacked by 1041 species of insects, 82 species of nematodes and more than 40 diseases has been reported from different parts of the world [2]. Nearly, 250 insect and mites, 380 fungal pathogens have been found to attack aerial part of tea. Amongst these, 167 insect pests and 190 fungal pathogens have reported from North East India [3]. Pests and diseases attack various parts of the plant like leaf, stem, root, flowers and seeds resulting more than 1015% yield loss. In Assam, organic tea production is increasing year after year. Management and control of pests and diseases rely on the application of synthetic pesticides [4,5]. Due to indiscriminate use of chemical pesticides many problems have arised like resistance, pest resurgence, environmental pollution and risks to human health. That is why pesticides could not be considered as the primary solution to curb the pest problem. Hence considerable efforts have been directed toward the use of bio–control agents as an alternative to the use of chemical pesticides. Biological control is an important, effective, ecofriendly and economical component of Integrated Pest Management (IPM) in almost all important crops for development of sustainable cropping systems. Of the various bio-control agents considered, the entomopathogenic fungi like Metarhizium anisopliae (Metschnikoff) Sorokin and Beauveria bassiana (Bals.) Vuill have received considerable attentions as a viable alternative to chemical pesticides because the micro climate of tea garden is much favourable to develop and survive the entomopathogenic fungi that affects various types of insect pests. At field application rates, they are considered safe to non–target hosts. The fungi can be mass-produced relatively easily on artificial substrates and can be applied under a wide range of environmental conditions [6]. M. anisopliae, is also known as green muscardine fungi, is one of the highly promising frontline fungal bio-agent recognized for their biological control potential against arthropods. Metarhizium species are known to attack a wide range of arthropods, more than 200 species of insects in over 50 families including termites. It is currently being used as a biological insecticide to control a number of pests such as termites, thrips, aphids etc [7].
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Citation: Dutta P, Kaushik H, Singh H, Bhuyan RP, Nath DJ, et al. (2017) Effect of Climatic Parameters on Persistence of Metarhizium anisopliae. Vegetos 30:2.
doi:10.4172/2229-4473.1000236 Fungal persistence is an important area to consider for at least two reasons, firstly, it is desirable for the fungus to have a limited time span to minimize its possible harmful effects on non-target species. On the other hand, the fungus should persist for as long as possible to reduce the need for continuous spraying [8]. Several studies have shown that M. anisopliae can survive for days, months or even years in the field. However, this persistence depends largely on the environmental conditions of each area and the fungal isolate used [8-12]. Hence, the extrapolation of results to other regions is not straight forward. It is crucial to determine the persistence of M. anisopliae in the area where it is going to be used or using regularly. Precise knowledge of the amount of fungal spores that remain viable and effective will enable managers and/or farmers or growers to take appropriate measures in response to population increases or declines at any given time. To date, however, its persistence and viability after spraying over crop fields have not been determined. Therefore, the present study was undertaken with the objectives: to determine the persistence and viability of Metarhizium anisopliae in the tea ecosystem of Assam; to establish correlation between cfu counts of M. anisopliae with recorded climatic parameters and to study the role of M. anisopliae on biological and physico-chemical properties of soil.
Materials and Method Study area The experiment was conducted in two tea ecosystem. One was organic named Banaspaty Tea Estate located in Karbi-Anglong district of Assam, India. Its geographical coordinates are 26°14.777'N and 0.93°50.711'E with an elevation of 543 ft. Another one was inorganic tea ecosystem i.e., Experimental Garden for Plantation Crops (EGPC), Department of Tea Husbandry and Technology, AAU, Jorhat, Assam, India which is lying between longitude 26°42.975'N and latitude 094°11.805'E at an elevation 89 ft. 1600m2 under both the tea ecosystem was used for the present experiment. Climatic data throughout the period of study was collected from Department of Agricultural Meteorology, AAU, Jorhat and Regional Agricultural Research Station (RARS), Karbi-Anglong, Diphu.
Fungal spraying Monospores of M. anisopliae were used in this study. The culture was derived from the culture bank maintained under Mycology Research Section, Department of Plant Pathology, Assam Agricultural University, Jorhat, Assam, India. The same culture was identified by Indian Type Culture Collection (ITCC), Indian Agricultural Research Institute, New Delhi. For initial establishment of the fungus in the study area, three sprays at an interval of 30 days were done with liquid formulation of M. anisopliae at a concentration of 1X106 spore/ml of water. Spraying was done in the afternoon hours i.e. after 3 p.m. with the help of knapsack backpack sprayer (Aspee Bakpak Sprayer) with a capacity of 13 litres in such a way that the soil and tea bushes were wetted well. Before spraying the whole experimental area was irrigated with sprinkler irrigation for an hour so that soil surface gets atleast 20% of moisture.
Samples Soil, air and leaf samples were collected from the study area throughout the period of 1 year and 2 months at an interval of 30 days of the study to quantify the amount of viable colonies of M. anisopliae in each substrate. Volume 30 • Issue 2 • 1000236
Soil sampling On each sampling date approximately 250 g of soil was collected from 15 cm and 30 cm depth from the sprayed area from four different spots located at eight different directions of the experimental plot. Soil was collected from as near as possible to the plant roots without damaging them because the highest concentrations of the fungus occur in this area [13]. Sterile metallic spoons were used for collection of soil sample and collected individually in sterile plastic bags for transfer to the laboratory. In the laboratory, collected samples were divided into two equal parts and one part was mixed thoroughly to make a composite sample. This was again divided into four equal parts of which two parts were discarded. Left out parts of soil was again divided into four parts and again two parts were rejected as earlier. This process was repeated until 10 g (approx.) of soil was left. Each soil sample was then put in sterile plastic bags and stored in refrigerator at 4°C until analysis.
CFU quantification in soil samples One gram of each soil sample was suspended in 9 ml of sterile water, stirred in a vortex for 3 min. This mixture was then serially diluted to achieve dilutions of 10-1 to 10-6. These dilutions were replicated thrice separately and were used for analysis. Aliquot (1 ml) from each dilution and was later placed in petridishes containing Metarhizium specific medium [14] and incubated at 28 ± 1°C for 5 days. Developing colonies with general characteristics of the genus Metarhizum (colour, conidial size, conidial shape, etc) were grouped. M. anisopliae colonies were quantified to determine the number of cfu g-1 of soil.
CFU quantification in air samples To determine the viability of M. anisopliae (cfu’s in air), the spores were tried to trap by exposing petridishes containing Metarhizium specific media above the canopy of tea bushes. After 3 hours, exposed petridishes were sealed with parafilm and were taken to the laboratory for incubation at 28 ± 1°C for 5 days. Developing colonies with general characteristics of the genus Metarhizum (colour, conidial size, conidial shape, etc.) were grouped. M. anisopliae colonies were quantified to determine the number of cfu of air sample.
Plant sampling Leaf samples were taken from each plot as defined by the intersection of concentric circles and transects laid out at 5 m intervals in eight opposing directions, arranged on north, northeast, east, southeast, south, southwest, west, and northwest axes . Samples were cut with a sterile scissor and put individually in sterile plastic bags for transfer to the laboratory and stored in refrigerator at 4°C until analysis.
CFU quantification in vegetation Each piece of leaf was vigorously vortexed for 1 minute in 10 ml of sterile water. Aliquots (1 ml) of the suspension were serially diluted (10-1, 10-2, and 10-3) and plated on Metarhizium specific medium in petridishes and finally incubated at 28 ± 1°C for 5 days. These dilutions were replicated thrice and were used for analysis. Colonies developed with general characteristics of the genus Metarhizum (colour, conidial size, conidial shape, etc.) were grouped. M. anisopliae colonies were quantified to determine the number of cfu g-1 of leaf. • Page 2 of 8 •
Citation: Dutta P, Kaushik H, Singh H, Bhuyan RP, Nath DJ, et al. (2017) Effect of Climatic Parameters on Persistence of Metarhizium anisopliae. Vegetos 30:2.
doi:10.4172/2229-4473.1000236
Estimation of soil biological and physico-chemical properties Soil samples of the treated plot were collected both prior and after the application (towards the end of the study period) of the entomopathogen so as to estimate the following soil parameters. Estimation of soil pH: pH of the soil was determined in 1:25 soil water suspension using a glass electrode pH meter (Eutech Instruments, pH 700) [15]. Estimation of soil organic carbon: Organic carbon was determined by wet digestion method given by Walkley and Black [16]. One gram of soil was weighed into a 500 ml erlenmeyer flask and 10 ml of 1N potassium dichromate solution was added to it. Then 20 ml of concentrated sulfuric acid was added and mixed by gentle rotation for 1 minute, taking care to avoid throwing soil up onto the sides of the flask. It was then allowed to stand for 30 minutes for completion of the reaction. 200 ml of deionized water was added to dilute the solution. On addition of 10 ml of orthophosphoric acid the solution was heated up, so it was allowed to cool down. Finally, 1 ml of diphenylamine indicator was added and back titrated the mixture with 0.5N FAS solution until the colour changes from violet through blue to bright green. One set of blank was also prepared and titrated in the same manner (instead of soil 1 ml of water was taken). The result was expressed in percentage. Estimation of microbial biomass carbon: MBC was determined by chloroform fumigation extraction technique of Vance et al. [17]. 10 g of soil in a 50 ml glass beaker was placed in a dessicator (Duran). In the dessicator another beaker was taken containing 100 ml ethanol free chloroform. Alongside another beaker was taken containing soda lime to absorb the excess moisture during fumigation. Dessicator was then closed. The air inside the dessicator was removed by opening the valve for few minutes. On reduction of pressure the chloroform boiled vigorously. The dessicator was then incubated in dark at 25 ± 5°C for 24 hours. After fumigation the chloroform was removed by repeated evacuation. The soil was then extracted with 25 ml 0.5M K2SO4 by oscillating at 200 rpm for 30 minutes and filtered through Whatman No. 42 filter paper. One set of control was prepared by extracting soil without fumigation. To 8ml of soil extract in a 100 ml erlenmeyer flask, 2 ml of 66.7 mM K2Cr2O7 and 15 ml of digestion mixture were added. The mixture was gently refluxed for 30 minutes, allowed to cool and diluted with 20 ml distilled water. The excess K2Cr2O7 was measured by back titration with FAS using phenanthroline ferrous sulphate complex solution as an indicator. The mbc was calculated from the differences in extractable organic carbon between the fumigated and non-fumigated soil and expressed as μg g-1 soil. Estimation of NPK: Total N in the compost was determined by modified Kjeldahl method [18]. Bray’s method [19] was followed for estimation of available phosphorus in soil. This procedure is primarily meant for soils that are moderately to strong acids (pH around 5.5 or less) and the K content was determined using Flame Photometer [15]. Estimation of exchangeable cations: Five (5) g of soil was weighed in a 150 ml erlenmeyer flask. Then 25 ml of neutral N NH4Ac solution was added to it. The contents of the erlenmeyer flask were shaked on an electric shaker for 5 minutes and was kept as such overnight and then filtered. The filtrate was feeded into the atomizer of the flame photometer (128 μc, Systronics). The reading was noted and the amount of exchangeable Ca++, Mg++, Na+ and K+ in the sample were calculated. Volume 30 • Issue 2 • 1000236
Result Persistence of M. anisopliae in organic and inorganic tea ecosystem Persistence of M. anisopliae in organic tea ecosystem: To study the persistence of M. anisopliae in the experimental area soil, leaf and air samples were collected both before and after the application of the entomopathogen and analysed for cfu counts. Soil samples of both the experimental area (organic and inorganic tea garden) showed no positive colonies of M. anisopliae prior to the application of the entomopathogen. When soil samples were collected during March, 2014 after a month from the first spray cfu of M. anisopliae was recorded as 9 x 106 and 8 x 106 cfu g-1 of soil at 15 cm and 30 cm soil depth, respectively (Table 1). Again when the soil samples were collected during the month of April, 2014 after the second spray (March, 2014) a decline in the population of the fungus was recorded (7 x 106 and 5 x 106 cfu g-1 soil at 15 and 30 cm soil depth, respectively). The population of the fungus was found to increase steadily after the third spray in April, 2014 till June, 2014 at 15 cm depth and to July, 2014 at 30 cm depth of soil. At 15 cm soil depth there was a slight decrease in the population of M. anisopliae during the month of July, 2014 which was immediately followed by increased population during the month of August, 2014. Similarly, during August, 2014 the fungal population was also decreased at 30 cm soil depth. The decline of the fungus was more abrupt in the month of November, 2014 for both the soil depth i.e., 2 ×106 and 4 ×106 cfu g-1 of soil for 15 cm and 30 cm depth of soil, respectively (Table 1). Subsequently, a sharp increasing trend in the population of the applied entomopathogen was recorded till January, 2015 at 15 cm soil depth. While a decreasing trend was observed at 30 cm depth from December, 2014 till February, 2015. Observations recorded displayed that the entomopathogen persisted and remained viable in both 15 cm and 30 cm depth of soil in the sprayed area throughout the study period i.e., 1 year and a month. Similarly, in air sample M. anisopliae was found to persist for 12 months in the sprayed area with negative results in between. Air borne spores of the entomopathogen were detected in Metarhizium specific petridishes on March, 2014 when exposed for three hours in the experimental area and decreased to zero a month later till October, 2014. Population of M. anisopliae was found to increase gradually from November, 2014 to January, 2015 with a cfu count of 32 and 65 cfu per air samples, respectively. There was a slight decrease in the population of aerospore of M. anisopliae during February, 2015 (53 cfu per air samples) which further decreased to zero as no M. anisopliae colonies were detected in the air (Table 1). No positive M. anisopliae colonies were detected as epiphyte in the vegetative cover of the sprayed area during the whole period of study. Persistence of M. anisopliae in inorganic tea ecosystem: Similarly in EGPC samples were taken for analysis prior to the application of the entomopathogen and showed no cfu counts of M. anisopliae in the soil, vegetation and air samples. After the first spray in the month of February, 2014 cfu counts at 15 cm and 30 cm depth of soil was 6 ×103 and 5 ×103 cfu g-1of soil respectively for the month of March, 2014 (Table 2). When the samples were collected a month later (April, 2014) from the second spray (March, 2014) an increase in the cfu of M. anisopliae was observed at 15 cm soil depth but was found to decrease at 30 cm soil depth. After the third application of the entomopathogen during April, 2014; the cfu count at 15 cm depth of soil showed an increasing trend from May, 2014 to September, 2014 with a cfu count of 15 ×103 and 24 ×103 cfu g-1 of soil, respectively. These values however fluctuated in between. cfu count of M. anisopliae tended to • Page 3 of 8 •
Citation: Dutta P, Kaushik H, Singh H, Bhuyan RP, Nath DJ, et al. (2017) Effect of Climatic Parameters on Persistence of Metarhizium anisopliae. Vegetos 30:2.
doi:10.4172/2229-4473.1000236 Table 1: cfu of M. anisopliae in organic tea ecosystem. 15 cm soil depth*
30 cm soil depth*
Months
cfu (x 106) g-1 soil
Log cfu
cfu (x 106) g-1 soil
Log cfu
Feb, 14
0.0
-
0.0
-
0.0
Mar, 14
9.0
6.95
8.0
6.90
27.0
Apr, 14
7.0
6.84
5.0
6.69
0.0
May,14
13.0
7.11
9.0
6.95
0.0
June, 14
25.0
7.39
13.0
7.11
0.0
July, 14
22.0
7.34
25.0
7.39
0.0
Aug, 14
27.0
7.43
14.0
7.14
0.0
Sept, 14
24.0
7.38
22.0
7.34
0.0
Oct, 14
4.0
6.60
9.0
6.95
0.0
Nov, 14
2.0
6.30
4.0
6.60
32.0
Dec, 14
18.0
7.25
6.0
6.77
60.0
Jan, 15
27.0
7.43
5.0
6.69
65.0
Feb, 15
13.0
7.11
3.0
6.47
53.0
Mar, 15
8.0
6.90
4.0
6.60
0.0
g-1 soil
Air Samples**
g-1 soil
* Data are mean of 3 replications ** Data are mean of 5 replications Table 2: cfu of M. anisopliae in inorganic tea ecosystem. 15 cm soil depth*
30 cm soil depth*
Months
cfu (x 103) g-1 soil
Log cfu
Feb, 14
0.0
Mar, 14
6.0
Apr, 14
Air Samples**
cfu (x 103) g-1 soil
Log cfu
-
0.0
-
0.0
3.77
5.0
3.69
19.0
12.0
4.07
3.0
3.47
0.0
May,14
15.0
4.17
11.0
4.04
1.0
June, 14
14.0
4.14
21.0
4.32
0.0
July, 14
15.0
4.17
20.0
4.30
0.0
Aug, 14
17.0
4.23
14.0
4.14
0.0
Sept, 14
24.0
4.38
15.0
4.17
0.0
Oct, 14
9.0
3.95
13.0
4.11
0.0
Nov, 14
7.0
3.84
5.0
3.69
26.0
Dec, 14
7.0
3.84
4.0
3.60
36.0
Jan, 15
5.0
3.69
3.0
3.47
59.0
Feb, 15
3.0
3.47
6.0
3.77
38.0
Mar, 15
6.0
3.77
14.0
4.14
42.0
g-1 soil
decline thereafter from October, 2014 until a lowest of 3 ×103 cfu g-1 of soil was achieved during February, 2015. Towards the end of the study period there was a slight increase in the population of M. anisopliae at 15 cm soil depth i.e. 6 ×103 cfu g-1 of soil. From the third application of M. anisopliae to the end of the sampling period, population of M. anisopliae at 30 cm soil depth fluctuated widely showing a highest cfu of 21 x 103 cfu g-1 of soil during the month of June, 2014. From July, 2014 the cfu count of of M. anisopliae at 30 cm soil depth appeared to decline till January, 2015 with a cfu count of 20 ×103 and 3 ×103 cfu g-1 of soil, respectively. Population of the entomapathogen was found to increase for the month of February and March, 2015. The decline of the fungal population was more abrupt in the airborne spores than in the soil. A decline from 19 cfu to 0 cfu of M. anisopliae occurred with a brief recovery of 1 cfu in the month of May, 2014. Subsequently fungal population started to increase from November, 2014 onwards until a highest of 59 cfu was obtained in the month of January, 2015 (Table 2). There was slight downfall in the population thereafter in the month of February, 2015 about it was found to recover during March, 2015. M. anisopliae persisted for 13 months in soil and air of the sprayed area with positive and negative results in between. Volume 30 • Issue 2 • 1000236
g-1 soil
Further, no positive colonies of M. anisopliae were found in the analysed sample of leaves collected before and after the application of the microbial agent on the vegetative cover of inorganic garden.
Correlation of cfu of M. anisopliae with climatic parameters Correlation of cfu of M. anisopliae with climatic parameters for organic tea ecosystem: Table 3 showed the correlation between recorded climatic parameters and cfu count of M. anisopliae in the soil and air samples. Positively non-significant correlation was found between the cfu count of M. anisopliae at both the soil depth i.e. 15 cm and 30 cm and maximum temperature. This means that with increase in maximum temperature population of M. anisopliae at both the soil depth also increases but it was not found to be statistically significant. Similarly, cfu count at 15 cm depth of soil was found to have positively non-significant correlation with minimum temperature. Significantly, a positive correlation at 1% level of significance was found between cfu count at 30 cm depth of soil and minimum temperature i.e. on increase of minimum temperature population of M. anisopliae in the respective soil samples also increases. Correlation was found significantly positive between cfu count of both the soil depth and • Page 4 of 8 •
Citation: Dutta P, Kaushik H, Singh H, Bhuyan RP, Nath DJ, et al. (2017) Effect of Climatic Parameters on Persistence of Metarhizium anisopliae. Vegetos 30:2.
doi:10.4172/2229-4473.1000236 Table 3: Correlation of cfu of M. anisopliae with climatic parameters for organic tea ecosystem. Maximum temperature
Minimum temperature
Relative humidity morning
Relative humidity evening
Total rainfall
BSSH
No. of rainy days
cfu counts at 30 cm
0.526NS
0.793**
0.660*
0.821**
0.918**
-0.589*
0.862**
cfu counts at 15 cm
0.016NS
0.393NS
0.445NS
0.631*
0.555*
-0.861**
0.624*
cfu counts at air
-0.858**
-0.744**
-0.093NS
-0.296NS
-0.492NS
0.005NS
-0.634*
Note: NS=non significant correlation, *=Significant at 5% level of significance, **=Significant at 1% level of significance
relative humidity (RH) of evening hours. Positively non-significant correlation was found between cfu counts at 15 cm soil depth and morning RH but the correlation was found to be positively significant between cfu counts at 30 cm soil depth and morning RH at 5% level of significance. Similarly, significantly positive correlations were found between the cfu of M. anisopliae in the soil samples of both the depth and the variables like: total rainfall and number of rainy days. Increase in bright sun shine hours decreases the cfu counts of M. anisopliae in the soil sample as correlation between them was found to be negatively significant (Table 3). However, significantly negative correlation was found between the airborne spores, temperature (minimum and maximum) and number of rainy days. This means that with the decrease in minimum and maximum temperature and number of rainy days, the population of airborne spores of M. anisopliae increases. Correlation study between cfu counts in air with relative humidity, total rainfall and bright sun shine hours showed non-significant. Correlation of cfu of M. anisopliae with climatic parameters for inorganic tea ecosystem: Correlation of cfu counts of M. anisopliae at 15 cm soil depth was found positively significant with maximum and minimum temperature, evening relative humidity, total rainfall and number of rainy days (Table 4). While cfu counts at 15 cm soil depth had non-significant correlation with morning relative humidity, evaporation and wind speed. Data presented in Table 4 showed that increase in bright sun shine hours decreases the cfu counts of M. anisopliae at 15 cm soil depth and vice versa. Significantly, positive correlation was found between the cfu counts at 30 cm soil depth and the variables like: temperature (maximum and minimum), evening relative humidity, total rainfall and number of rainy days at 1% level of significance. Table 4 showed that morning relative humidity, evaporation and wind speed had positively non-significant correlation with cfu counts of M. anisopliae at 30 cm soil depth. Similarly, with the increase in bright sun shine hours cfu counts of M. anisopliae decreases at 30 cm soil depth and vice versa. Again it was observed that population of airborne spores increase with decrease in the variables like: maximum and minimum temperature, evening relative humidity, total rainfall and number of rainy days. However, correlation of cfu counts in air was found to be non-significant with morning relative humidity, evaporation, wind speed and bright sun shine hours (Table 4).
Estimation of soil biological and physico-chemical properties of organic and inorganic tea ecosystem Estimation of soil biological and physico-chemical properties of organic tea ecosystem: Soil samples of both the depth (i.e. 15 cm and 30 cm) were collected before as well after the application of the entomopathogen to estimate pH, available nitrogen (as N kg ha-1), available phosphorus (as P2O5 kg ha-1), available potash (as K2O kg ha1 ), exchangeable cations like Ca++, Mg++, Na+ and K+ {Cmol (P+) kg-1}, organic carbon (%) and microbial biomass carbon (μg g-1 soil). Table 5 showed that when samples were collected before application of M. Volume 30 • Issue 2 • 1000236
anisopliae, concentration of available macronutrients (N, P2O5 and K2O) and exchangeable cations (except Mg++) were more in the upper profile (15 cm) than in the lower profile (30 cm) of soil. Similarly, organic carbon at 15 cm depth of soil was 0.31% but it was lower at 30 cm soil depth with 0.27% organic carbon. Microbial biomass carbon was also found to be more in 15 cm soil depth (335.00 μg g-1 soil) than in 30 cm soil depth (164.00 μg g-1 soil). The pH at 30 cm soil depth was found to be more acidic (pH of 4.62) than 15 cm depth of soil (pH of 4.80). When soil samples were collected towards the end of the study period during March, 2015 there was a change in all the studied soil parameters. Available N at 15 cm and 30 cm soil depth increased from 459.00 and 438.00 N kg ha-1 to 470.60 and 450.60 N kg ha-1, respectively. Similarly available phosphorus and potash were also found to increase in the treated plot. Except Ca++ other three cations (Mg++, Na+ and K+) were also increased in the soil (Table 5). After the application of M. anisopliae organic carbon was increased from 0.31% and 0.27% to 0.59% and 0.34% at 15 cm and 30 cm soil depth, respectively. Similarly, microbial biomass carbon had also increased to 1987.81 and 1500.92 μg g-1 soil at 15 cm and 30 cm soil depth, respectively. There was a slight variation in the pH of the experimental plot after the application of the entomopathogen as it increased to 4.88 and 4.70 at 15 cm and 30 cm soil depth, respectively. Estimation of soil biological and physico-chemical properties of inorganic tea ecosystem: Similar to column 5.3.1, soil samples were collected both before and after the application of M. anisopliae to estimate the soil biological and physico-chemical properties. pH of the soil before application of the entomopathogen was found 4.19 and 4.25 at 15 cm and 30 cm soil depth respectively (Table 5). Concentration of available N, P2O5 and K2O were more at 15 cm than at 30 cm depth of soil. Similarly, the concentration of exchangeable cations (Ca++, Mg++, Na+ and K+), organic carbon and microbial biomass carbon were also comparatively higher at 15 cm soil depth (Table 5). Soil samples collected after the application of M. anisopliae during March, 2015 showed increased in soil biological and physicochemical properties like pH, available macronutrients (N, P2O5 and K2O) and exchangeable cations (except Ca++). pH of the treated plot was found to increase from 4.19 and 4.25 to 4.37 and 4.45 at 15 cm and 30 cm soil depth respectively. When samples were analysed for organic carbon, we found increase of organic carbon from 0.15% and 0.10 % to 0.28% and 0.13% at 15 cm and 30 cm soil depth respectively. Similarly, microbial biomass carbon was also increased after the application of M. anisopliae from 180.71 and 154.70 μg g-1 soil to 1700.55 and 1459.35 μg g-1 soil at 15 cm and 30 cm soil depth, respectively.
Discussion The result of the present study on persistence indicated that M. anisopliae remained viable and detectable for 1 year and a month in both soil and air in inorganic tea ecosystem. While it was detectable for 1 year and a month in soil and for approximately 12 months in air in organic tea ecosystem with negative periods in between. GuerreroGuerra et al. [20] from Mexico reported that M. acridum can survive • Page 5 of 8 •
Citation: Dutta P, Kaushik H, Singh H, Bhuyan RP, Nath DJ, et al. (2017) Effect of Climatic Parameters on Persistence of Metarhizium anisopliae. Vegetos 30:2.
doi:10.4172/2229-4473.1000236 Table 4: Correlation of cfu of M. anisopliae with climatic parameters for inorganic tea ecosystem. Maximum temperature
Minimum temperature
Relative humidity morning
Relative humidity evening
Total rainfall Evaporation
Wind speed
BSSH
No. of rainy days
cfu counts at 30 cm
0.833**
0.825**
0.023NS
0.721**
0.849**
0.508NS
0.247NS
-0.630*
0.779**
cfu counts at 15 cm
0.790**
0.874**
0.064NS
0.732**
0.799**
0.443NS
0.305NS
-0.604*
0.815**
cfu counts at air
-0.672**
-0.765**
0.113
-0.537*
-0.638*
-0.411
-0.414
0.428
-0.612*
NS
NS
NS
NS
Note: NS=non significant correlation, *=Significant at 5% level of significance, **=Significant at 1% level of significance Table 5: Soil biological and physico-chemical properties of organic and inorganic tea ecosystem. Soil Depth
parameters
Available (kg ha-1) pH
N
Exchangeable Cations {Cmol (P+) kg-1}
P2O5
K 2O
Ca++
Mg++
Na+
K+
Organic Carbon (%)
Microbial Biomass Carbon (μg g-1 soil).
Organic tea ecosystem Before application of M. anisopliae 15 cm
4.80
459.00
36.00
305.22
0.80
0.55
1.46
0.47
0.31
335.00
30 cm
4.62
438.00
32.00
294.47
0.80
0.60
1.29
0.49
0.27
164.00
After application of M. anisopliae 15 cm
4.88
470.60
41.00
320.50
0.30
0.70
1.60
0.79
0.59
1987.81
30 cm
4.70
450.60
35.40
307.80
0.70
0.65
1.46
0.89
0.34
1500.92
Inorganic tea ecosystem Before application of M. anisopliae 15 cm
4.19
446.00
33.90
225.79
0.80
0.50
1.38
0.40
0.15
180.71
30 cm
4.25
421.00
29.60
214.63
0.60
0.40
1.35
0.37
0.10
154.70
After application of M. anisopliae 15 cm
4.37
468.80
36.50
240.20
0.50
0.90
2.15
0.62
0.28
1700.55
30 cm
4.45
440.90
34.60
232.40
0.40
0.60
2.00
0.54
0.13
1459.35
for 1 year and 4 months in soil and for 8 months on plant leaves and in the air. This result is also similar to those obtained by Bruck [21] who reported that inoculated conidia of M. anisopliae persist up to 1 year in the rhizosphere. In the soil environment the hypocrealen entomopathogenic fungi can persist; however its population build up greatly relies on the conversion of host cadaver resources into infective conidia that are released from the cadavers over time on sporulation [22] as the entomopathogen need resources to grow. The cfu count of M. anisopliae in soil was found to be higher than those of air which may be due to the agricultural practices like pruning, weeding, manuring etc. During the period of our study we observed negative result in the air from April, 2014 to October, 2014 in both the tea ecosystem. From November, 2014 there was a sudden increase in the population of M. anisopliae of air sample. In tea gardens of Assam pruning, manuring, skiffing and other agricultural activities were done during the month of November-December which may induce the release of conidia from soil and plant surface into the air as observed by Guerrero-Guerra et al. [20]. Decreased population with increased soil depth may be due to the effect of solar radiation that increases the soil temperature at increasing soil depth which ultimately reduces the conidial viability. Little information is available on fungal persistence in the vegetative cover more particularly for entomopathogens. Moore et al. [23] reported that the half-life of conidia in treated vegetation was 4.3 days while, Hunter et al. [24] reported that M. acridum persisted for almost 7 days in the vegetation of agricultural areas in Australia. Guerrero-Guerra et al. [20] asserted that the initial concentrations of M. acridum in the vegetative cover of Mexico declined steadily until its complete disappearance after 8 months. The result of the present study differ from those of the above mentioned studies as M. anisopliae did not persist in the treated vegetative cover of both the tea Volume 30 • Issue 2 • 1000236
ecosystem even for a month after its application. This result is similar to those obtained by Braga et al. [25] who evaluated the persistence of fungal conidia in the vegetative cover and reported its persistence nearly low to negative because ultraviolet solar radiation caused its inactivation over a period of hours. If the conidia are exposed directly to solar radiation then its survival is substantially reduced [26] as solar radiation increases the temperature at the leaf surface, which in turn reduces the humidity around the leaf. As a consequence, the surface tension that binds conidia to the leaf decreases, causing their release [27] and also splashes of rain water wash the epiphyte [28] because of which no fungal population was observed on leaf surfaces. If conidia of the entomopathogens are applied on the abaxial surface of leaves [29] its persistence can be enhanced as they will be safe from direct exposure to solar radiation and splashes of rain water. However, the effectiveness of this method will depend on the behaviour of the target insect pest. Understanding the influence of environmental conditions on transmission of M. anisopliae and predicting where and when cycling of the fungus is likely to occur, requires the knowledge of the persistence within the biotic/abiotic environment [30]. Highest (27 x 103) and lowest (2 x 103) cfu g-1 of soil at 15 cm soil depth of organic tea ecosystem was obtained at 28.8°C with 85.5% RH and 22.0°C with 75.5% RH respectively while at 30 cm soil depth highest (25 x 103) and lowest (3 x 103) cfu g-1 of soil was at 29.3°C with 85.5% RH and 18.8°C with 67.5% RH, respectively. However, in inorganic tea ecosystem highest (24 x 103) cfu g-1 of soil at 15 cm soil depth was obtained at 29.1°C with 87.1% RH while at 30 cm soil depth highest cfu g-1 (21x103) was recorded at 29.2°C. On the other hand lowest cfu g-1 was recorded as 3 x 103 during February, 2015 at 15 cm soil depth and during April, 2014 and January, 2015 at 30cm soil depth. The recorded temperature was 19.2°C with 76% RH during February, • Page 6 of 8 •
Citation: Dutta P, Kaushik H, Singh H, Bhuyan RP, Nath DJ, et al. (2017) Effect of Climatic Parameters on Persistence of Metarhizium anisopliae. Vegetos 30:2.
doi:10.4172/2229-4473.1000236 2015. Average temperature of 25.1°C with 68.5% RH and 17.9°C with 76% RH was recorded during the month of April, 2014 and January, 2015, respectively. Zimmerman [8] asserted that M. anisopliae is a mesophilic fungus with a temperature range generally between 15 to 35°C and the optimum for germination and growth lies between 25 and 30°C. Our result as mentioned above revealed that M. anisopliae remained viable and detectable for 1 year and a month in both soil and air in inorganic tea ecosystem. While it was detectable for 1 year and a month in soil and for approximately 12 months in air in organic tea ecosystem with negative periods in between. This seems logical because collected weather data showed that the average monthly temperature approximately lies between 17 to 30°C. Considerable variability exists among genotypes in their thermal characteristics. For instance, Fargues et al. [31] found that four isolates of M. anisopliae var. acridum equally induced 98–100% mortality in the desert locust at 25 and 30°C, and none at 40°C, but there were significant differences among the isolates at 35°C, with mortalities ranging from 40 to 100%. During the month of April, 2014 and January, 2015 though the temperature was within the growth range of M. anisopliae but it could not withstand the lower humidity so the conidial population was low. In a report Daoust and Roberts [32] recorded that conidia of M. anisopliae survive better at moderate temperatures when relative humidity is as high as 85%. Humidity is a very important environmental factor not only affecting the efficacy but also the survival of an entomopathogen. Generally, a high relative humidity is necessary for germination of M. anisopliae.
with very low organic matter content hinder the population of the entomopathogens. This may be the result of higher cation exchange capacities in soil with greater organic matter enhance adsorption of fungal conidia or because soils with greater organic matter also have greater diversity and density of arthropod hosts in which the fungi can multiply [42-44]. The increase in biological and physico-chemical properties in the soil observed in the present study may or may not be statistically significant so for further confirmation this work has to be carried out for another 5-10 years [45].
Although climatic parameters have been reported as important abiotic factors influencing the persistence of entomopathogenic fungi but in the present study efforts were also made to observe the effect of M. anisopliae on soil biological and physico-chemical properties. Soil pH of the experimental plots (i.e. organic and inorganic tea ecosystem) was found to be acidic both before and after application of M. anisopliae. This seems logical since fungi in general are more tolerant to acidity than to alkalinity [33] and studies says that M. anisopliae is better adapted to slightly acidic soils [34,35]. Similar, observation was also reported by Asensio et al. [36] from Alicante (SE Spain). After the application of M. anisopliae, we observed increased in soil physico-chemical properties (except Ca++) may be due to the fact that cell wall of aerial conidia of entomopathogens is composed of a great diversity of carbohydrates that contribute to the formation of hydrogen bonds between the conidia and hydrophobic and hydrophilic surfaces [37,38]. This may be involved in the interaction of conidia with the clay surfaces for ion exchange as previously reported for Histoplasma capsulatum (Ascomycota: Onygenales) [39]. During the study period, after 1 year of application of M. anisopliae increased in available soluble nutrient content was observed. This was not in agreement with the earlier work of Guerrero-Guerra et al. [20] who did not find any correlation between the number of cfu g-1 of soil and the soil physicochemical parameters recorded. Organic carbon content of the treated plot had increased towards the end of the study period after the application of M. anisopliae (from 0.31% and 0.27% organic carbon to 0.59% and 0.34% organic carbon at 15 cm and 30 cm soil depth respectively in organic tea ecosystem and from 0.15% and 0.10% organic carbon to 0.28% and 0.13% organic carbon respectively in inorganic tea ecosystem). Similarly, microbial biomass carbon in both the tea ecosystem was found to increase after 1 year of application of M. anisopliae. M. anisopliae being a soil fungus have a filamentous growth habit and thereby is a major component of the soil microbial biomass and their biomass can exceed that of crop roots [40]. The ratio of MBC to total organic carbon provides a measure of organic matter dynamics [1,8,33]. Mietkiewski et al. [41] reported that soils
References
Volume 30 • Issue 2 • 1000236
Conclusion The results of this study displayed that the population of M. anisopliae has the potential of accumulating in the soil and air under the proper environmental conditions. Conidia present in these areas decay after one year so, fungal spraying must be done accordingly to achieve a better control. We could not able to detect positive colonies of M. anisopliae as an epiphyte in the vegetative cover of the study area. If conidia are applied on the abaxial surface of leaves, persistence of the entomopathogen could be enhanced on phylloplanes. The degree to which the targeting of abaxial leaf surfaces will be efficacious will also depend on the behaviour of the target insect pest. The results of this study also indicated that the availability of M. anisopliae conidia in the soil effect the properties of the soil. But the mechanism is still to be explored [46,47].
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Citation: Dutta P, Kaushik H, Singh H, Bhuyan RP, Nath DJ, et al. (2017) Effect of Climatic Parameters on Persistence of Metarhizium anisopliae. Vegetos 30:2.
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Volume 30 • Issue 2 • 1000236
Author Affiliations
Top
1 Department of Plant Pathology, Assam Agricultural University, Jorhat-785013, Assam, India 2 Department of Entomology, Gochar Mahavidyalaya, Saharnapur-247451, U.P., India 3 Department of Tea Husbandry and Technology, Assam Agricultural University, Jorhat-785013, Assam, India 4 Department of Soil Science, Assam Agricultural University, Jorhat-785013, Assam, India
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