Ethnobotanical Leaflets 12: 698-704. 2008.

 

 

Compatibility of Metarhizium anisopliae (Metsch.) Sorok. with Ocimum sanctum Linn. (Tulsi) (Lamiaceae) Extracts

 

J. Francis Borgio*, B. Jesvin Bency1 and Neha Sharma2

 

*PG and Research Department of Microbiology, St. Joseph’s College (Autonomous),

Bangalore - 560027, India.

E. mail: borgiomicro@gmail.com

1Department of Microbiology, Malankara Catholic College, Mariagiri,

Kaliakavilai - 629153, India.

E. mail: jesvinbency@yahoo.co.in

2Bioengineering and Research Centre (BERC), Rai Foundation College,

Raipur, Chattisgarh, India.

 

Issued 12 September 2008

 

Abstract

            The compatibility of Metarhizium anisopliae with Ocimum sanctum was studied in vitro. Leaves, roots, stems and seed extracts of O. sanctum were mixed in a Potato Dextrose Agar and Potato Dextrose Broth. M. anisopliae was inoculated and the mycelial dry weight and spore count were assessed. The behavior of the fungus with the extracts was similar in terms of mycelial dry weight, except for methanol extracts of leaves, ether extracts of roots, water and acetone extracts of seeds and benzene, methanol and acetone extracts of stems which reduced the mycelial dry weight of the fungal colonies. Benzene extract of leaves and methanol extract of roots of O. sanctum were found to be highly compatible with M. anisopliae whereas ether extract of roots and benzene as well as acetone extracts of stem were classified as very toxic. The results of the current study revealed that O. sanctum extracts did not affect the inoculum potential of M. anisopliae in terms of mycelial dry weight and spore count and hence M. anisopliae was compatible with O. sanctum. 

 

Key Words: Compatibility, Metarhizium anisopliae, Ocimum sanctum, biological control.

 

Introduction

         The production of food requests for methods that use non-chemical inputs for pest control to reduce harmful side-effects of pesticides on public health and the environment. In such production systems, pests are essentially managed by biological agents, which are considered as important factors in insect population reduction. Entomopathogenic fungus, Metarhizium anisopliae (Metsch.) Sorokin (Deuteromycotina: Hyphomycetes) is an important natural control agent for several pests (Borgio and Sahayaraj, 2007).  Botanical pesticides are also used extensively in most agroecosystems to control insect pests. Ocimum sanctum (holy basil), called Tulsi in India, is ubiquitous in Indian tradition. The crude alcoholic extracts had shown antifeedant activities on Jute semilooper, Anomis sabulifera (Malik and Rafique, 1989), and mosquito repellent and toxic properties (Batta and Santhakumari, 1970; Deshmukh et al., 1982); the essential oil had exhibited larvicidal activity against C. quinquefasciatus, A. aegypti and A. stephensi (Pathak et al., 2000). With the recent increase in the use of O. sanctum plant extracts in integrated pest management systems, where the entomopathogenic fungi M. anisopliae are also used, a study on the compatibility among them become dessessive for combined use.

Plant extracts used in agriculture might affect the action of entomopathogenic fungi in the same way, as do the chemical pesticides. The use of incompatible plant extracts may inhibit the development and reproduction of these pathogens, affecting pest control (Malo 1993, Duarte et al. 1992, Anderson and Roberts 1983). On the other hand, the use of selective products is an important strategy in IPM (Integrated Pest Management). In some cases, compatible products may be associated with entomopathogenic fungi, increasing control efficiency (Moino and Alves 1998; Quintela and McCoy 1998).

            Reports were not available on the compatibility of O. sanctum extracts with M. anisopliae. The present study deals with the compatibility of leaves, roots, stems and seed extracts of indigenous O. sanctum with M. anisopliae in-vitro.

 

Materials and methods

Microorganism tested

            Pure culture of Metarhizium anisopliae NCIM 1311 was obtained from the National Collection of Industrial Microorganism (NCIM), National Chemical Laboratory, Pune, India.

 

Culture media

            M. anisopliae were cultured in potato dextrose agar (PDA) and potato dextrose broth (PDB) medium (HiMedia, Mumbai, India).

 

Preparation of plant extract

            Ocimum sanctum was collected from Rajgurunagar of Pune, Maharashtra, India in March 2008. About 200g of O. sanctum leaves were ground in mortar and pestle with 10ml of ether. Resulting paste was collected in a conical flask and allowed to mix properly by placing in a shaker at 200 rpm for 24 hours. Then the paste was filtered through double-layered muslin cloth and the filtrate of leaves in water was used for further studies. Similar protocol was adapted to prepare leaf extracts using benzene, methanol, acetone and water. The same procedure was used to prepare the extract from other plant parts like roots, stems and seeds. All the extracts were stored at 4oC for further analysis.

 

Mycelial dry weight assessment

            Five mm disc of 72 hours old M. anisopliae was inoculated into sterile PDB. 500 l of plant extract (0.05%) was added into seeded PDB and incubated at room temperature for 7 days. Controls were amended with respective solvents. After a week, the mycelial mat was taken with sterile spatula, placed in sterile dishes containing filter paper. The initial weight of the paper was recorded. The Petri dishes were kept in hot air oven at 50°C for 30 minutes and the final weight of the fungal mat along with the filter paper was recorded immediately. The difference between the final and initial weight was considered as dry weight of mycelium.

 

Spore production assessment

            500 l of plant extract (0.05%) was added to sterile PDA at 45± 50 C and poured into sterile Petri dishes. After solidification of the medium, M. anisopliae was inoculated and incubated at room temperature for 7 days. After incubation, a central colony disk (5mm) was placed in a test tube and the conidia were suspended in 10 ml of sterile water containing 0.02% Tween 20 and quantified using a Neubauer chamber.

 

Statistical analysis

Data were subjected to descriptive statistics using STATISTICA/w 5.0.

 

Compatibility calculation: Compatibility was calculated according to Alves et al. (1998), as follows:

 

2 0 (MDW) + 8 0 (SC)

T  =    ____________________________

          100

 

   In this model, values for vegetative growth (MDW) and sporulation (SC) are given in relation to the control (100%). Where T= 0 to 30 = very toxic; 31 to 45 = toxic; 46 to 60 = moderately toxic; 60 - 90 =compatible; > 90 = highly compatible.

 

Results and Discussion

Leaves, roots, stems and seed extracts of   O. sanctum did not affect conidial production regardless of the ether, benzene, methanol, acetone and water solvents used (Table 1). The behavior of the fungus with the extracts was similar in terms of mycelial dry weight, except for methanol extracts of leaves, ether extracts of roots, water and acetone extracts of seeds and benzene, methanol and acetone extracts of stems which reduced the mycelial dry weight of the fungal colonies (Table 1).

 

Table 1 Mycelial dry weight (mean±SD) and spore count (mean±SD) of Metarhizium anisopliae NCIM 1311 with different solvent extracts of various parts of Ocimum sanctum.

 

Name of the plant part

Solvent

MDW in gm

(mean ± SE )

SC in conidia/ml (mean ± SE )

 

 

Leaves

Ether

0.0607 ± 0.001

1.80x109 ± 5.75x104

Benzene

0.0772 ± 0.013

1.77x109 ± 5.86x103

Methanol

0.0472 ± 0.013

6.15x108 ± 2.29x104

Water

0.0695 ± 0.004

9.34x108 ± 1.14x103

Acetone

0.0212 ± 0.041

5.32 x108 ± 2.29x102

 

 

Roots

Ether

0.0433 ± 0.003

3.64x108 ± 3.25x104

Benzene

0.0624 ± 0.021

4.69x108 ± 1.87x 104

Methanol

0.0734 ± 0.005

9.57x108 ± 3.18x103

Water

0.0803 ± 0.021

1.02×109± 2.06x103

Acetone

0.0510 ± 0.013

1.10x109 ± 2.10x104

 

 

Seeds

Ether

0.0603±0.0012

1.82×109± 1.07x 104

Benzene

0.0549±0.0045

1.41×109± 2.14x104

Methanol

0.0643±0.0011

4.15×108± 2.26x104

Water

0.0448±0.0017

1.03×109± 1.43x103

Acetone

0.0446±0.0024

5.06×108± 1.48x103

 

 

Stems

Ether

0.0626 ± 0.005

1.42x109 ± 7.49x103

Benzene

0.0494 ± 0.013

2.09x108 ± 2.97x104

Methanol

0.0458 ± 0.005

7.40x108± 2.40x103

Water

0.0626 ± 0.004

8.04x108 ± 3.87x102

Acetone

0.0470 ± 0.020

5.08x108 ± 1.77x103

 

 

Control

 

Ether

0.0802 ± 0.006

3.11x109± 2.88x102

Benzene

0.0799 ± 0.003

1.87x109 ± 3.01x104

Methanol

0.0812 ± 0.001

1.05x109± 4.45x103

Water

0.0839 ± 0.007

3.77x109 ± 3.88x104

Acetone

0.0842 ± 0.002

2.57x109 ± 2.26x104

 

MDW - Mycelial dry weight, SC – Spore count

 

When the data concerning mycelial dry weight and spore count were submitted to the formula for the determination of T (Table 2), benzene extract of leaves and methanol extract of roots were found to be highly compatible; ether extract of leaves, ether and benzene extracts of seeds and methanol extracts of stems were compatible (Table 2). Ether extract of roots and benzene as well as acetone extracts of stem were classified as very toxic for M. anisopliae (Table 2).

 

Table 2 "T" values and compatibility classification of various parts of Ocimum sanctum with different solvent extracts on Metarhizium anisopliae NCIM 1311.

 

Name of the

plant part

Solvent

Values of "T"1

Classification2

 

 

Leaves

Ether

61.44

C

Benzene

95.04

HC

Methanol

58.47

MT

Water

36.37

T

Acetone

31.59

T

 

 

Roots

Ether

20.16

VT

Benzene

35.67

T

Methanol

90.98

HC

Water

41.58

T

Acetone

46.35

MT

 

 

Seeds

Ether

61.84

C

Benzene

74.06

C

Methanol

47.44

MT

Water

32.52

T

Acetone

26.34

VT

 

 

Stems

Ether

52.13

MT

Benzene

21.30

VT

Methanol

67.67

C

Water

31.98

T

Acetone

26.97

VT

 

1Alves et al. (1998); 2 HC = highly compatible, C = compatible, MT = moderately toxic,

T = toxic, VT = very toxic.

  

The formula proposed by Alves et al. (1998) represented in an appropriate way the toxic effect on the entomopathogenic fungi in vitro. Thus, when the treatment is compatible in vitro, there are strong evidences of its selectivity under field conditions. However, a high toxicity in vitro does not always mean that the same will happen in the field (Alves et al. 1998), but rather indicates only the possibility of the occurrence of damage of this nature. In addition, under field conditions, vegetative growth inhibition may not be a good indication of fungicidal effects such as spore viability (Loria et al. 1983). Under field condition, compatibility germination should be considered as the most important factor (Malo 1993; Anderson and Roberts 1983) due to the fact that pathogens infect insects through conidia germination by ingestion or contact. The survival of inoculum of the entomopathogenic fungi in the field is made by conidia. In the beginning of the epizootic, the conidia are responsible for the first disease focuses (Alves and Lecuona, 1998).

Information about compatibility among M. anisopliae and plant extracts used in pest control is scarce. Aguda et al. (1986) and Gonzalez et al. (1996) verified the negative effect caused by neem on M. anisopliae germination and conidiogenesis. According to the model of Alves et al. (1998), neem oil was moderately toxic for M. anisopliae. However, compatibility of chemical pesticides with this mycopathogen have been studied by Fargues (1975) and Anderson et al. (1989), Mohammed et al. (1987); Castinerias et al. (1991). Hassan and Charnely (1989) revealed the in-consistent interaction between fungus and insecticides. Li and Holdam (1994) observed chlorinated hydrocarbon insecticides as more deleterious than other insecticide groups to the mycopathogen. They observed extremely detrimental effect of chlorpyriphos, tempephos and malathion to mycelial growth and sporulation of M. anisopliae, while carbamate insecticides like carbofuran, methomyl and oxamyl were moderately toxic.

The results of the current study revealed that O. sanctum extracts did not affect the inoculum potential of M. anisopliae in terms of mycelial dry weight and spore count and hence M. anisopliae was compatible with O. sanctum. Field studies with the application of O. sanctum and M. anisopliae together with pests could provide extra information to that obtained by this study to help in the development of IPM strategies in agriculture.

 

References

1.      Aguda RM. Rombach MC. and Shepard BM. 1986. Effect of "neem" oil on germination and sporulation of the entomogenous fungus Metarhizium anisopliae. Int. Rice Res. Newsletter; 11: 34-35.

2.      Alves SB. and Lecuona RE. 1998. Epizootiologia aplicada ao controle microbiano de insetos. In- Controle microbiano de insetos, ed. S. B. Alves. Fealq, São Paulo; pp 97-170.

3.      Alves SB. Moino A. and Almeida JEM. 1998. Produtos fitossanitários e entomopatógenos. In- Controle microbiano de insetos, ed. S.B. Alves. Fealq, São Paulo; pp.217-238.

  1. Anderson TE. and Roberts DW. 1983. Compatibility of Beauveria bassiana Isolates with Insecticide Formulations Used in Colorado Potato Beetle (Coleoptera: Chrysomelidae) Control. J. Econ. Entomol; 76: 1437-1441.

5.      Anderson TE. Hajek AE. Roberts DW. Preisler K. and Robertson JL. 1989. Colorado potato beetle (Coleoptera: Chrysomelidae) : effects of combinations of Beauveria bassiana with insecticides. J. Econ. Entomol.; 82 : 83-89.

  1. Batta SK. and Santhakumari G. 1970. The antifertility effect of Ocimum sanctum and Hibiscus rosa sinensis. Indian J Med Res; 59: 777781.
  2. Borgio JF. and Sahayaraj K. 2007. Bioefficacy of the entomopathogenic fungus, Metarhizium anisopliae (Metsch.) Sorokin (Deuteromycotina: Hyphomycetes) on Dysdercus cingulatus (Fb.) eggs. In: Proceedings of the National Seminar on Technology and Management of Bioresearches. Eds. M. Narayann, T. Sethuramalingam, K. Sahayaraj; 29–34.

8.      Castinerias A. Calderon A. and Lopez A. 1991. Effect of biocides and fertilizers used in banana cultivation in Cuba on entomopathogenic fungi, Metarhizium anisopliae. Proteccion-de Plantas; 1 : 33-42.

  1. Deshmukh PB. Chavan SR. and Renapurkar DM. 1982. A study of insecticidal activity of twenty indigenous plants. Pesticides; 12: 710.
  2. Duarte A. Menendez JM. and Triguero N. 1992. Estudio preliminar sobre la compatibilidad de Metarhizium anisopliae com algunos plaguicidas quimicos. Rev. Baracoa; 22: 31-39.
  3. Fargue T. 1975. Etude experimental dans la nature de Beauveria and Metarhizium a dose reduite contre Lephinotarsa decemlineata. Annals of Zoology Ecology and Animals; 7: 247-264.

12.  Gonzalez DME. Valbuena PBF. Rivera MA. Bustillo PAE. and Chaves B. 1996. Viabilidad del hongo Metarhizium anisopliae en mezcla con agroquimicos, Rev. Colomb. de Entomol; 22: 31-36.

  1. Hassan AEM. and Charnely AK. 1989. Ultrastructural study of the penetration by Metarhizium anisopliae through dimilin affected cuticle of Manduca sexta. J.  Invertebr. Pathol; 54: 117-124.

14.  Li DP. and Holdom DG. 1995. Effects of nutrients on colony formation, growth, and sporulation of Metarhizium anisopliae (Deuteromycotina, Hyphomycetes). J.  Invertebr. Pathol; 65: 253-260.

15.  Loria R. Galaini S. and Roberts DW. 1983. Survival of inoculum of the Entomopathogenic Fungus Beauveria bassiana as Influenced by Fungicides. Environ. Entomol; 12: 1724-1726.

  1. Malik MS. and Rafique M. 1989. Effects of methanol extracts of Ocimum sanctum Linn. on jute semilooper. Indian J Entomol; 51: 8489.
  2. Malo AR. 1993. Estudio sobre la compatibilidad del hongo Beauveria bassiana (Bals.) Vuill. con formulaciones comerciales de fungicidas e insecticidas. Rev. Colomb. de Entomol; 19; 151-158.
  3. Mohammed AKA. Joann PP. and Nelson FRS. 1987. Compatibility of Metarhizium anisopliae var. anisopliae with chemical pesticides. Mycopathologia; 99: 99-105.
  4. Moino Jr A. and Alves SB. 1998. Efeito de Imidacloprid e Fipronil sobre Beauveria bassiana (Bals.) Vuill. E Metarhizium anisopliae (Metsch.) Sorok. e no Comportamento de Limpeza de Heterotermes tenuis (Hagen). An. Soc. Entomol. Brasil; 27: 611-619.

20.  Pathak N. Mittal PK. Singh OP. Sagar V. and Vasudevan P. 2000. Larvicidal action of essential oils from plants against the vector mosquitoes Anopheles stephensi (Liston) Culex quinquefasciatus (Say) and Aedes aegypti (L). Int Pest Control; 42:53.

21.  Quintela ED. and McCoy CW. 1998. Synergistic Effect of Imidacloprid and Two Entomopathogenic Fungi on the Behavior and Survival of Larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae) in Soil. J. Econ. Entomol; 91: 110-122.