OPCE-STR.vp Acta Bot. Croat. 73 (1), 37–50, 2014 CODEN: ABCRA 25 ISSN 0365-0588 eISSN 1847-8476 Influence of crop species and edaphic factors on the distribution and abundance of Trichoderma in Alfisol soils of southern India VELLAISAMY MUNIAPPAN, THANGAVELU MUTHUKUMAR* Root and Soil Biology Laboratory, Department of Botany, Bharathiar University, Coimbatore 641 046, Tamilnadu, India Abstract – The effect of crop species and edaphic factors on the distribution of Tricho- derma species in Alfisol soil under different agrosystems was evaluated. Each soil sample was assayed for nine abiotic factors and culturable microfungal populations. Fungal abun- dance was determined by dilution plate technique, and the identification of fungi was based on morphological characteristics. Pearson’s correlation coefficient was used to de- termine the relationship of association between these factors and the presence and abun- dance of Trichoderma species in each soil type. The abundance of soil fungi ranged be- tween 7.0 × 103 and 13.6 × 103 colony forming units (cfus) per gram of dry soil. The population densities of the two Trichoderma species (T. koningii and T. viride) isolated in the present study varied significantly with crop species and their abundance (varied from 0.6 to 3.6 × 103 cfus g–1 dry soil). Twenty-two other colony-forming fungal types with an abundance ranging between 7.0 × 103 and 13.6 × 103 cfus g–1 dry soil were also isolated in the present study. As soil pH negatively influenced relative abundance of T. koningii, soil P and relative abundance of T. viride were significantly and positively correlated to each other. Further, relative abundance of T. koningii was significantly and positively correlated to relative abundance of Aspergillus fumigatus but negatively correlated to relative abun- dance of Stachybotrys atra. Likewise, a significant negative correlation existed between relative abundance of T. viride and Absidia glauca. Key words: Alfisol soil, crops, edaphic factors, fungal abundance, soil pH, Trichoderma Introduction Trichoderma are abundant free living soil fungi that are highly interactive in root, soil and foliar environments. Their significance resides in their potential for control of soil borne plant pathogens. Trichoderma act as biocontrol agents through competition for nutrients or space, mycoparasitism and antibiosis (WELLS 2000). Some strains also act as plant growth promoters through solubilization and sequestration of organic nutrients (HARMAN et al. ACTA BOT. CROAT. 73 (1), 2014 37 * Corresponding author, e-mail: tmkum@yahoo.com Copyright® 2014 by Acta Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved. 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:56 Color profile: Generic CMYK printer profile Composite Default screen 2004a). There is a reasonable amount of information about the behavior of many soil borne plant pathogens in the soil environment. However, little attention has been given to factors affecting growth of antagonistic organisms such as Trichoderma in the soil (WAKELIN et al. 1999). Factors affecting the growth of Trichoderma under laboratory conditions have been extensively studied, but the correlation between various growth responses in in situ and in vivo conditions are dubious (COOK and BAKER 1983, PAPAVIZAS 1985). This limited knowl- edge on the ecology of these biocontrol agents coupled with the environment variability limits the success of Trichoderma as a biological control agent under field situations (LEWIS and PAPAVIZAS 1991). There is an urgent need for more information on the factors affecting the distribution of Trichoderma, in agro ecosystems. The Alfisol soils that constitute around 10% of the ice-free land area are the dominant soil types in many tropical and subtropical areas, including peninsular India (RUST 1983). Alfisols are characterized by high fertility and water-holding capacity, moderate leaching and have at least 35% base saturation (SOIL SURVEY STAFF 1994). Therefore, extensive areas of Alfisols have been used for cultivation or forestry (RAO et al. 1991). As high yields and sustainable agriculture are targeted by farmers, researchers, politicians and society, knowl- edge of the fungal population present in the soil and their functions in the corresponding ecosystems is vital. Although some soil fungi (e.g., mycorrhizal fungi) are intensively stud- ied, little is known about fungal community structures and community dynamics in agricul- tural soils. It is generally agreed that only a small percentage of the 1.5 million fungi world-wide are culturable (HAWKSWORTH and ROSSMAN 1997). Nevertheless, up to now, many studies have investigated fungal diversity by using culture–dependent approaches (ATTITALLA et al. 2012, OKOTH et al. 2009, SARIAH et al. 2005). In spite of studies examining the bioinoculant potential of Trichoderma against various plant pathogens in Alfisol soils (MANJULA et al. 2004, KUMAR et al. 2012), information on the survivability and proliferation of fungi in relation to soil type and factors is limited. The ability of Trichoderma to dispose and to colonize the rhizosphere will determine its effectiveness as a biocontrol agent. Thus, an understanding of the quantitative distribution of the fungus in different agro-ecosystems is essential before it can be developed into biological formulations for field application. The following study was undertaken with the objectives of (i) quantifying the populations of Trichoderma spp., from cultivated ecosystems, (ii) to determine if crop species had any sig- nificant influence on the distribution or abundance Trichoderma spp. and (iii) to character- ize the influence of edaphic factors and co-occurring fungal species on populations of Trichoderma. Materials and methods Sampling Alfisol soil samples were collected from agrosystems within a 2 km radius of Coim- batore (11°04’ N and 76°92’ E, altitude 426.7–550 m a.s.l.), Tamil Nadu, India. These in- cluded soil samples under ten different crop species: tomato (Lycopersicon esculentum, soil 1), coconut (Cocus nucifera, soil 2), sorghum (Sorghum bicolor, soil 3), ground nut (Arachis hypogea, soil 4), maize (Zea mays, soil 5), sugarcane (Saccharum officinarum, soil 6), rice (Oryza sativa, soil 7), turmeric (Curcuma longa, soil 8), areca nut (Areca catechu, soil 9) and papaya (Carca papaya, soil 10). 38 ACTA BOT. CROAT. 73 (1), 2014 MUNIAPPAN V., MUTHUKUMAR T. 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:56 Color profile: Generic CMYK printer profile Composite Default screen Ten undistributed soil cores measuring 3 cm diameter were randomly taken from the depth of 0–15 cm of the top horizon adjacent to the plants, and combined. The composite samples were placed in airtight sterile Ziploc polythene bags and transported to the labora- tory. Half of the soil samples used to assess the soil microfungal populations was stored at 4 °C, while the other half was air dried, passed through a 2-mm screen to remove coarse de- bris and plant materials, and used to analyze the physico-chemical properties of the soil. Determination of soil characteristics The composite soil sample from each agrosystem was divided into five equal parts and assessed for various properties. One hundred mL of sterile distilled water was added to 100 g of soil sample, thus yielding a 1:1 soil suspension. The samples were stirred briefly and al- lowed to equilibrate for 1h. The pH of each sample was then measured using a digital pH meter (ELICO LI 610, Hyderabad, India) by immersing the electrode in the sediment at the bottom of the soil slurry. One hundred mL of distilled water was added to 10 g of dry soil to make a suspension of 1: 10 (W/V) dilution. The electrical conductivity EC was measured using a digital EC TDS analyzer (ELICO CM 183, Hyderabad, India). The total nitrogen (N) and phosphorus (P) in the soils were determined by micro-Kjeldahl and molybdenum blue methods respectively (JACKSON 1971). Soil K was estimated by the flame photometric method after extraction in ammonium acetate solution (JACKSON 1971). Other mineral (Fe, Zn, Cu and Mn) element composition was also analyzed according to JACKSON (1971). Evaluation of soil micro fungal population Soil dilution technique was used to evaluate the total number of fungal colony forming units (cfus) in the soil samples and to isolate pure cultures (DANIELSON and DAVEY 1973). Each composite soil intended for the assessment of micro fungal populations was divided into five equal parts. Five grams (on dry weight basis) of the composite soil subsample were added to 250 mL sterile conical flasks containing 100 mL of sterilized distilled water. Flasks were shaken vigorously for five minutes. One mL aliquots of the 1:103 dilutions were spread evenly on each of the five Petri dishes (100 ´ 15 mm) containing 20 ml of agar rose Bengal medium (MARTIN 1950) supplemented with 0.06 g L–1 of streptomycin sulphate for each composite soil subsample. Petri plates were incubated at room temperature (26–30 °C) till the development of fungal colonies. After four days, all fungal colonies were counted and the plates were incubated again for another three days. Many colonizers sporulated after seven days, so at that time individual colony types were counted, including those of Trichoderma. For each of the colony types counted, representative colonies were isolated and brought under pure culture for identification. Results were recorded as the number of colonies per plate. Averages of composite soil subsamples were used to determine the abundance and frequency of soil micro fungal populations. Semi-permanent slides were prepared by mounting fungi in lactophenol containing 0.5% cotton blue. The characterization of Trichoderma isolates into species aggregates were based on cultural and morphological characters (BISSETT 1984, 1991, 1992; BISSETT et al. 2003; CHAVERRI et al. 2001, 2004; CHAVERRI and SAMUELS 2002, 2003; RIFAI 1969; WEBSTER 1964; WEBSTER and RIFAI 1968). The other co-existing soil fungi were also identi- ACTA BOT. CROAT. 73 (1), 2014 39 INFLUENCE OF CROP SPECIES AND EDAPHIC FACTORS ON TRICHODERMA 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:56 Color profile: Generic CMYK printer profile Composite Default screen fied according to the descriptions by BARRON (1972), DOMSCH et al. (1980), ELLIS (1971), NELSON et al. (1983), PITT (1979), and SUBRAMANIAN (1970). Abundance (isolates of a species as percent of total isolates) and frequency of occur- rence (percent from total plate samples) for each soil was calculated using the formulae: Abundance = Number of colonies of a particular fungus in the sample ´ 100 Total number of fungal colonies of the sample Frequency = Number of agrosystems in which a particular fungus was present ´ 100 Total number of agrosystems examined Statistical analysis Data on soil characteristics and microfungal populations were subjected to Analysis of Variance and the means were separated using Duncan’s Multiple Range Test (DMRT) (SPSS for windows, Version 9). The relationships between soil factors and microfungal populations and among fungal populations were analyzed by Pearson’s correlation. Data were log transformed prior to statistical analysis. Results Soil factors Measurement of pH, EC, soil N, P, K, Fe, Zn, Mn, Cu and Fe are presented in table 1. The soils in the present study were near neutral to alkaline with a pH range of 7.23 to 8.23. The EC ranged from 0.245 to 1.05 dSm–1. The soils were nutrient deficient with various nu- 40 ACTA BOT. CROAT. 73 (1), 2014 MUNIAPPAN V., MUTHUKUMAR T. Tab. 1. Characteristics of the Alfisol soils used in the study. Soil pH EC (dS m–1) Soil nutrients (mg kg–1) Nitrogen Phospho- rus Potassium Copper Iron Zinc Manga- nese S1 7.23 a 0.245 a 9.8 b 0.25 a 42.00 c 1.41 a 26.44 f 1.24 d 8.24 c S2 7.64 b 0.636 f 10.1 b 0.75 b 50.00 c 1.31 a 10.42 d 1.52 f 2.84 a S3 7.93 c 0.299 b 14.0 e 1.15 c 40.00 c 1.21 a 5.44 a 1.28 de 4.24 b S4 7.89 c 0.536 e 11.5 c 0.26 a 51.00 d 1.80 b 8.32 c 0.86 b 8.22 c S5 8.23 e 0.315 b 6.4 a 0.28 a 42.00 c 1.24 a 6.78 b 0.76 ab 4.24 b S6 8.10 d 0.302 b 14.0 e 0.45 a 18.00 a 3.64 e 10.60 d 1.48 f 18.60 e S7 7.74 b 1.050 g 12.6 d 1.20 c 51.00 d 2.82 c 8.50 c 1.08 c 7.06 c S8 8.12 d 0.408 c 10.4 b 1.55 d 31.00 b 4.06 f 14.80 e 0.64 f 15.56 d S9 7.99 c 0.448 d 10.6 b 1.30 c 19.00 a 3.08 d 9.41 cd 1.40 ef 16.20 d S10 7.93 c 0.548 e 14.0 e 1.35 c 51.00 d 4.24 f 10.36 d 1.60 f 20.24 f Means in a column followed by same letter(s) are not significantly (p > 0.05) different according to Duncan’s multiple range test. Soil types S1–S10 are explained in Methods. 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:56 Color profile: Generic CMYK printer profile Composite Default screen A C T A B O T .C R O A T .73 (1),2014 41 IN F L U E N C E O F C R O P S P E C IE S A N D E D A P H IC F A C T O R S O N T R IC H O D E R M A Tab. 2. Distribution and frequency (percent from total plate samples) of soil microfungi associated with different crop species. Soil types S1 – S10 are ex- plained in Methods. Fungal species Soils Frequency (%) S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Absidia glauca Hagem + + + + + + + 70 Aspergillus candidus Link ex. Fr. + + + + + + + 70 Aspergillus flavipes (Bain. et Sart.) Thom et Chruch + + + + + + + 70 Aspergillus fumigatus Fresen. + + + + + + 60 Aspergillus sp. + + + + + 50 Aspergillus pulverulentus (McAlpine) Thom. + + + + + + 60 Aspergillus terreus Thom. + + + + + + + + + 90 Cladosporium cladosporioides (Fres.) de Vries. + + + + + + 60 Curvularia lunata (Wakker) Boedijn. + + + + 40 Drechslera halodes (Drechsler) Subram. et B.L. Jain + + + + + + 60 Mucor sp. + + + + + + 60 Fusarium coeruleum (Lib.) Sacc. + + + + + 50 Fusarium solani (Mart.) Sacc. + + + + + + + 70 Humicola grisea Traeen + + + + + 50 Nigrospora sphaerica (Sacc.) E.W. Mason + + + + 40 Penicillium adametzi Zaleski + + + + + + 60 Penicillium citrinum Sopp + + + + + 50 Penicillium rubrum Stoll + + + + + + + + 80 Rhizopus stolonifer (Ehrenb.) Vuill. + + + + + + + + 80 Scopulariopsis brevicaulis (Sacc.) Bainier + + + + + + 60 Stachybotrys atra Corda. + + + + + + + + 80 Trichoderma koningii Oudem + + + + + + + + 80 Trichoderma viride Pers. ex. Fries + + + + + + + + + 90 Verticillium albo-atrum Reinke et Berth + + + + 40 7 4 1 M u n i a p p a n a n d M u t h u k u m a r . p s U : \ A C T A B O T A N I C A \ A c t a - B o t a n 1 - 1 4 \ 7 4 1 M u n i a p p a n a n d M u t h u k u m a r . v p 1 9 . o u j a k 2 0 1 4 1 6 : 4 6 : 5 6 C o l o r p r o f i l e : G e n e r i c C M Y K p r i n t e r p r o f i l e C o m p o s i t e D e f a u l t s c r e e n trient levels ranging as follows: 6.4 to 14.0 mg Kg–1 of total N, 0.25 to 1.55 mg Kg–1 of total P, 18 to 51 mg Kg–1 of exchangeable K, 5.44 to 26.44 mg Kg–1 of Fe, 0.60 to 1.60 mg Kg–1 of Zn, 2.84 to 20.24 mg Kg–1 of Mn and 1.21 to 4.24 mg Kg–1 of Cu. Trichoderma populations Trichoderma was isolated from all the Alfisol soils (Tab. 2). These include Trichoderma koningii Oudem and Trichoderma viride Pers. ex. Fries. The average population density of T. koningii ranged between 0.6 × 103 cfus and 3.6 × 103 cfus g–1 of soil, T. koningii was not found in S. bicolor and C. papaya-cultivated soils. Trichoderma viride average populations ranged between 0.6 × 103 cfus and 1.6 × 103 cfus g–1 of soil. Trichoderma viride was absent from A. hypogea soil. Populations of both T. koningii (F7,36 = 2.45, p < 0.05) and T. viride (F8,36 = 2.31, p < 0.05) significantly varied with plant species. Based on the overall occur- rence in different soils, T. viride was the most frequent Trichoderma species occurring in 90% of the soil samples examined. Co-occurring microfungal population The 22 colony-forming soil microfungi, apart from T. koningii and T. viride were classi- fied into 20 species belonging to 14 genera and two could not be identified to species level (Tab. 2). Total fungal colonies significantly varied among crop species (F9,40 = 4.485, p < 0.001) and were maximum in S. bicolor soil (14 × 103 cfu g–1 soil) and minimum in S. officinarum soil (7 × 103 cfu g–1 of soil) (Fig.1). Aspergillus terreus and T. viride occurred in the soils of most crop species examined, whereas Nigrospora sphaerica and Verticillium albo-atrum were present in a minimum number of soil samples (Tab. 2). Among the 10 soil samples examined Trichoderma species (T. koningii) was abundant only in three soils (A. hypogea, C. nucifera, and L. esculentum soils) (Tab. 3, Fig. 2). Relative abundance of other soils was shared by Aspergillus terreus (S. officinarum, A. catechu and C. papaya soils), Stachybotrys atra (S. bicolor soil), Drechslera halodes (C. longa soil), Mucor sp. (Z. mays soil) and Rhizopus stolonifer (O. sativa soil). Aspergillus was the most diverse genus in the present study with six species followed by Penicillium (three species). 42 ACTA BOT. CROAT. 73 (1), 2014 MUNIAPPAN V., MUTHUKUMAR T. c f f e b a d e c b 7 9 11 13 15 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Soils 0 T o ta l fu n g a l a b u n d a n c e (1 0 c fu g s o il ) 3 – 1 // Fig. 1. Total soil micro fungal populations in different agrosystems. Vertical bars indicate ± 1 S.E. Bars bearing same letter(s) are not significantly different according to Duncan’s multiple range test (p > 0.05). Soil types S1–S10 are explained in Methods. 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:57 Color profile: Generic CMYK printer profile Composite Default screen A C T A B O T .C R O A T .73 (1),2014 43 IN F L U E N C E O F C R O P S P E C IE S A N D E D A P H IC F A C T O R S O N T R IC H O D E R M A Tab. 3. Abundance of soil microfungal species (isolates as a percent of total isolates isolated from agrosystems of different crop species). Abundant species in each agrosystem is presented in bold. Soil types S1 – S10 are explained in Methods. Fungus name Soils S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Absidia glauca 4.00 8.82 1.67 2.86 1.82 1.67 4.88 Aspergillus candidus 6.00 2.94 2.86 2.86 5.00 2.00 4.88 Aspergillus flavipes 2.00 3.33 2.38 5.71 7.27 5.00 10.00 Aspergillus fumigatus 12.00 13.24 4.29 5.00 7.14 12.73 Aspergillus pulverulentus 4.41 7.14 1.82 6.67 2.00 2.44 Aspergillus sp. 6.67 5.71 1.82 6.67 6.00 Aspergillus terreus 14.00 17.65 8.57 16.67 17.14 9.09 5.00 20.00 17.07 Cladosporium cladosporioides 2.86 3.33 7.14 11.43 1.82 7.32 Curvularia lunata 1.47 2.86 2.38 2.44 Drechslera halodes 2.00 8.57 1.67 1.82 15.00 2.00 Fusarium coeruleum 1.47 4.76 2.86 1.67 4.00 Fusarium solani 2.00 11.43 10.00 7.14 8.33 14.00 14.63 Humicola grisea 2.00 6.67 5.71 1.67 6.00 Mucor sp. 1.47 7.14 16.67 5.45 3.33 4.88 Nigrospora sphaerica 2.94 2.86 5.00 4.76 Penicillium adametzi 18.00 1.47 8.33 2.38 3.64 5.00 Penicillium citrinum 1.47 2.38 8.57 2.00 4.88 Penicillium rubrum 2.00 5.88 14.29 3.33 2.86 5.45 5.00 4.88 Rhizopus stolonifer 4.00 2.94 1.67 2.86 14.55 6.67 8.00 7.32 Scopulariopsis brevicaulis 2.00 5.71 5.00 9.52 11.43 2.44 Stachybotrys atra 14.29 5.00 9.52 2.86 7.27 3.33 6.00 7.32 Trichoderma koningii 22.00 26.47 16.67 7.14 8.57 12.73 8.33 4.00 Trichoderma viride 8.00 4.41 11.43 9.52 8.57 12.73 11.67 12.00 12.20 Verticillium albo-atrum 2.94 2.86 2.00 2.44 7 4 1 M u n i a p p a n a n d M u t h u k u m a r . p s U : \ A C T A B O T A N I C A \ A c t a - B o t a n 1 - 1 4 \ 7 4 1 M u n i a p p a n a n d M u t h u k u m a r . v p 1 9 . o u j a k 2 0 1 4 1 6 : 4 6 : 5 7 C o l o r p r o f i l e : G e n e r i c C M Y K p r i n t e r p r o f i l e C o m p o s i t e D e f a u l t s c r e e n Relationship between soil factors and microfungal populations Correlation analysis revealed an absence of significant influence of soil factors on total soil microfungal populations. But abundance of T. koningii was significantly and negatively correlated to soil pH (r = –0.670; p < 0.05; n = 10) and relative abundance of T. viride was significantly and positively correlated to soil P (r = 0.697; p < 0.05; n = 10). Further, relative abundance of T. koningii was significantly and positively correlated to relative abundance of A. fumigatus (r = 0.749; p < 0.05; n = 10), but was significantly and negatively correlated to Stachybotrys atra (r = –0.766; p < 0.05; n = 10). Relative abundance of T. viride was signifi- cantly and negatively correlated to relative abundance of Absidia glauca (r = –0.727; p < 0.05; n = 10). Relative abundance of T. koningii and T. viride was significantly and nega- tively correlated to each other (r = –0.666; p < 0.05; n = 10). Among the co-occurring fungi, relative abundance of Penicillium adametzi was signifi- cantly and negatively correlated to soil pH (r = –0.712; p < 0.05; n = 10). In contrast, a signi- ficant positive correlation existed between soil EC and relative abundance of Rhizopus stolonifer (r = 0.756; p < 0.01; n = 10). As soil Cu was significantly and negatively correlated to relative abundance of Aspergillus fumigatus (r = –0.678; p < 0.05; n = 10), soil Fe was sig- nificantly and positively correlated to relative abundances of Aspergillus candidus (r = 0.713; p < 0.05; n = 10) and Penicillium adametzi (r = 0.826; p < 0.001; n = 10). The relative abundance of the unidentified Aspergillus sp. was significantly and posi- tively correlated to relative abundances of Aspergillus flavipes (r = 0.683; p < 0.05; n = 10) and Humicola grisea, (r = 0.817; p < 0.01; n = 10) and negatively to relative abundance of Curvularia lunata (r = –0.708; p < 0.01; n = 10). Likewise, relative abundance of Clado- sporium cladosporioides was significantly and positively correlated to relative abundances of Penicillium citrinum (r = 0.841; p < 0.01; n = 10) and Scopulariopsis brevicaulis (r = 44 ACTA BOT. CROAT. 73 (1), 2014 MUNIAPPAN V., MUTHUKUMAR T. 0% 20% 40% 60% 80% 100% S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Trichoderma koningii Trichoderma viride Other fungi Abundance S o il s Fig. 2. Percent abundance (%) of Trichoderma koningii and Trichoderma viride in relation to other soil fungi in different agrosystems. Soil types S1 – S10 are explained in Methods. 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:57 Color profile: Generic CMYK printer profile Composite Default screen 0.844; p < 0.001; n = 10). As the relative abundance of Curvularia lunata was significantly and positively correlated to the relative abundance of Mucor sp., (r = 0.669; p < 0.05; n = 10), it was significantly and negatively correlated to the relative abundance of Humicola grisea (r = –0.648; p < 0.05; n = 10). A significant positive correlation existed between rela- tive abundances of Mucor sp. and Stachybotrys atra (r = 0.637; p < 0.05; n = 10). In contrast, a significant negative correlation existed between the relative abundances of Rhizopus stolonifer and Scopulariopsis brevicaulis (r = –0.632; p < 0.05; n = 10). Discussion The frequent occurrence of Trichoderma species in Alfisol soils under different crop species is in accordance with the fact that species of Trichoderma are an abundant and bio- logically important component of soil micro-flora, though differences in their abundance can occur (SAMUELS 2006). In contrast, ATTITALLA et al. (2012) isolated Trichoderma har- zianum only from the five of the 23 soil samples they examined from Al-Jabal AL-Akhdar region of Libya. The population of Trichoderma in the present study varied from 0.6 to 3.6 × 103 cfus g–1 of soil, which is in agreement with published ranges. Nearly all temperate and tropical soils contain Trichoderma populations of 101 – 103 cfus g–1 of soil (see HARMAN et al. 2004a, b and references therein). Studies have shown that short-term changes in Trichoderma populations tend to occur in response to temporary changes in soil moisture, nutrient availability or soil temperature (KREDICS et al. 2003; WAKELIN et al. 1999). In the present study, populations of Trichoderma significantly varied with crop species. However, to our knowledge, there is only one previous study (OKOTH et al. 2009) that has examined the role of crop species on Trichoderma populations in addition to the substrate specificity of a few Trichoderma species. Nevertheless, the influence of plant species on Trichoderma has been mostly restricted to endophytic species. For example, Trichoderma stromaticum is found only in association with Theobroma species in tropical America (SAMUELS et al. 2000). It has been shown that plant species play a major role in determining the occurrence and distribution of fungal species (GRAYSTON et al. 1998; IBEKWE et al. 2002). In spite of their coexistence in 70% of the soil samples, populations of both the Trichoderma spp. were negatively related to each other. This suggests that both these soil fungi compete in the soil environment or the fungi may be reacting variedly to the given environmental conditions. The first view is supported by the fact that strains of T. viride are known to be highly com- petitive and are known to proliferate best in the presence of healthy plant roots, whereas, T. koningii tends to occur in diverse soil conditions (HJELJORD and TRONSMO 1998; SAMUELS 2006). The second view is supported by the fact that soil pH has been thought to be of great importance to the activity and occurrence of Trichoderma species (KREDICS et al. 2003, KÜÇÜK and KIVANÇ 2003, HARMAN et al. 2004a). KREDICS et al. (2003) showed that species of Trichoderma grow optimally around pH 4.0 to 5.0, and exhibit little or no growth below pH 2.0 or above pH 6.0. Nevertheless, the pH of the soils in the present study varied be- tween 7.23 and 8.23, and T. koningii was abundant in soils with a pH of 7.23 and 7.89. This corroborates the findings of GHERBAWY et al. (2004) reporting that Trichoderma was iso- lated from the Nile Valley soil in Egypt with a pH range of 7.4 to 8.4. In the present study, soil pH was found to have significant influence on the relative abundance of T. koningii but not T. viride. This clearly shows that even the taxa within a genus can respond differently to the same environmental factors. EASTBURN and BUTTER (1988) also reported that soil pH ACTA BOT. CROAT. 73 (1), 2014 45 INFLUENCE OF CROP SPECIES AND EDAPHIC FACTORS ON TRICHODERMA 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:57 Color profile: Generic CMYK printer profile Composite Default screen had no significant influence on the distribution of Trichoderma harzianum in a wide pH range of 6.2 to 7.9. Application of ammonia to the soil was found to increase the populations of Trichoderma spp. (SIMON et al. 1988). However, no significant relationship existed be- tween soil nutrient contents and populations of Trichoderma spp. indicating that the occur- rence of Trichoderma spp. is not strongly influenced by these soil nutrients, within the range encountered. This is in line with OKOTH et al. (2009) who also failed to find any relationship between Trichoderma spp., and soil nutrients in different land use types of Kenya. GHER- BAWY et al. (2004) also reported a lack of correlation between the abundance of Trichoderma to the physical and chemical properties of the soils or pH. It is therefore, possible to infer that the indigenous Trichoderma spp. in the present study might have adapted to the changes in the physical and chemical properties of these cultivated soils. Although there is reasonable information about the behavior of many soil fungi in their soil environment, little attention has been given to factors affecting the growth of Tricho- derma in field soils. Factors affecting growth of Trichoderma spp. on artificial cultured me- dia have been extensively studied, but the correlation between various growth responses on agar and in soil is dubious (COOK and BAKER 1983). Though most of the abiotic factors stud- ied did not significantly affect the abundance of Trichoderma spp. except soil pH and P, some biotic factors were found to be important in determining the distribution of both T. koningii and T. viride. Although the occurrence of T. koningii and T. viride was not signifi- cantly influenced by changes in the total fungal populations, the populations of the two Trichoderma spp. were associated with changes in certain fungal species like Absidia glauca, Aspergillus fumigatus and Stachybotrys atra. The existence of an inverse relation between Trichoderma spp. abundance and populations of Absidia glauca and Stachybotrys atra is interesting as these fungi may be competing with Trichoderma spp. for nutrients or space (CALVET et al. 1992). There have been relatively few studies examining the competi- tion of fungal species for nutrients, space or infection sites in the rhizosphere. Competition for carbon, N and Fe has been shown to be a mechanism associated with suppression of Fusarium wilt by Trichoderma species (WHIPPS 2001). Further, antibiotic production by isolates of Trichoderma / Gliocladium has been reported (HOWELL 1998). EASTBURN and BUTLER (1988) indicated species of Aspergillius (A. ustus, A. tamarii) and Penicillium (P. citrinum, P. chrysogenum, P. grieoroseum) to be positively related with a population of T. harzianum suggesting the tendency for the co-existence between these species. The relative abundance of T. koningii had a positive influence on the relative abundance of Aspergillius fumigatus in the present study. This positive relation between populations of T. koningii and A. fumigatus could be due to the lack of antagonism or synergism among these fungi or it could be an indirect response resulting from the similar response of the two organisms to certain environmental conditions. An understanding of the organisms occupying the same microsites as T. koningii and T. viride might give us insight into what types of habitats these Trichoderma spp. prefer. How- ever, the varied responses of T. koningii and T. viride to the same environmental factors clearly explain how a species within the same genus can respond diversely to similar envi- ronmental conditions. Unfortunately, there is little information available on the habitat re- quirements of the co-occurring species of Trichoderma, especially Aspergillus and Penicil- lium especially at microsite level. The frequent occurrence of the co-occurring A. fumigatus is in agreement with ATTITALLA et al. (2012) who also found Aspergillus spp. to be more fre- 46 ACTA BOT. CROAT. 73 (1), 2014 MUNIAPPAN V., MUTHUKUMAR T. 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:57 Color profile: Generic CMYK printer profile Composite Default screen quent in cultivated and non-cultivated soils of Libya. Interestingly, fast-growing Zygo- mycetes were not frequent in the present study. This corroborates the general observation that fast-growing Zygomycetes are not frequently isolated from agricultural soil in general, and from arable soils, in particular (HAGN et al. 2003). Trichoderma species are known to increase the concentration of a variety of nutrients under axenic conditions. However, the population of neither T. koningii nor T. viride was re- lated to any soil nutrients except soil P. Evidence does exist that Trichoderma species can solubilize various plant nutrients such as rock phosphate, Fe3+, Cu2+, Mn4+ and Zn, that can be unavailable to plants in certain soils (HARMAN et al. 2004a). Though there may be impor- tant abiotic or biotic factors, which were not accounted for in this study, the establishment and wide-spread occurrence of Trichoderma sp., appears to be more directly determined by the environment. A similar conclusion was reached by WIDDEN (1984, 1987) and EASTBURN and BUTLER (1988). They found that the environment had the greatest effect on the densities of several species of Trichoderma. Thus, it appears that in soils where the abiotic environ- ment is not greatly limiting, the factors of primary importance are those involving competi- tion, antagonism and synergism. In conclusion, our study clearly showed that crop species could influence the structure of the soil fungal communities. The results of the present study also indicated that the fungal populations in the studied soils are very stable and are not easily influenced by edaphic fac- tors. However, further studies involving a dual approach of culture-dependent and -inde- pendent techniques would throw more light on our understanding on the actual fungal diver- sity of the studied soils. Furthermore, such a study would also help us to deduce the influence of agricultural management practices on shifts in the indigenous fungal commu- nity structure. References ATTITALLA, I. H., ABDELRAWAF, S. S., OMAR, K. S., EL-KOMY, H. M. A., SARWAR, M., 2012: Occurrence and microbiological characteristics of Trichoderma in AL-Jabal Al-Akhdar region, Libya. Journal of Biological Sciences 12, 209–217. BARRON, G. L., 1972: The genera of Hypomycetes from soil. Robert E. Krieger Publishing Company, New York. BISSETT, J., 1984: A revision of the genus Trichoderma.I. section Longibrachiatnum sect. nov. Canadian Journal of Botany 62, 924–931. BISSETT, J., 1991: A revision of the genus Trichoderma. II. Intrageneric classification. Cana- dian Journal of Botany 60, 2357–2372. BISSETT, J., 1992; Trichoderma atroviride. Canadian Journal of Botany 70, 639–641. BISSETT, J., SZAKACS, G., NOLAN, C. A., DRUZHININA, I., GRADINGER, C., KUBICEK, C. P., 2003: New species of Trichoderma from Asia. Canadian Journal of Botany 81, 570– 586. CALVET, C., BAREA, J. M., PERA, J., 1992: In vitro interactions between the vesicular- -arbuscular mycorrhizal fungus Glomus mosseae and some saprophytic fungi isolated from organic substrates. Soil Biology and Biochemistry 24, 775–780. ACTA BOT. CROAT. 73 (1), 2014 47 INFLUENCE OF CROP SPECIES AND EDAPHIC FACTORS ON TRICHODERMA 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:57 Color profile: Generic CMYK printer profile Composite Default screen CHAVERRI, P., SAMUELS, G. J., 2002: Hypocrea lixii, the telemorph of Trichoderma harzi- anum. Mycological Progress 1, 283–286. CHAVERRI, P., SAMUELS, G. J., 2003 Hypocrea/Trichoderma (Ascomycota, Hypocreales, Hypocreaceae): Species with green ascospores. Studies in Mycology 48, 1–166. CHAVERRI, P., CANDOUSSAU, F., SAMUELS, G. J., 2004: Hypocera phyllostachydis and its Trichoderma anamorph, a new Bambusicolous species from France. Mycological Prog- ress 3, 19–28. CHAVERRI, P., SAMUELS, G. J., STEWART, E. L., 2001: Hypocera virens sp. nov., the teleo- morph of Trichoderma virens. Mycologia 93, 1113–1124. COOK, R. J., BAKER, K. F., 1983: The nature and practice of biological control of plant pathogens. St, Paul, American Phytopatological Society, New York. DANIELSON, R. M., DAVEY, C. B., 1973: The abundance of Trichoderma propagules and the distribution of species in forest soils. Soil Biology and Biochemistry 5, 485–494. DOMSCH, K. H., GAMS, W., ANDERSON, T. H., 1980: Compendium of soil fungi. Vol. 1 and 2, Academic Press, London. EASTBURN, D. M., BUTLER, E. E., 1988: Microhabitat characterization of Trichoderma har- zianum in natural soil: evaluation of factors affecting distribution. Soil Biology and Bio- chemistry 20, 547–553. ELLIS, M. B., 1971: Dematiaceous hyphomycetes. CAB Commonwealth Mycological Insti- tute, Kew, England. GHERBAWY, Y., DRUZHININA, I., SHABAN, G. M., WUCZKOWSKY, M., YASER, M., EL-NAGHY, M., PRILLINGER, H-J., KUBICEK, C. P., 2004: Trichoderma populations from alkaline ag- ricultural soil in the Nile valley, Egypt, consist of only two species. Mycological Prog- ress 3, 211–218. GRAYSTON, S. J., WANG, S., CAMPBELL, C. D., EDWARDS, A. C., 1998: Selective influence of plant species on microbial diversity in the rhizophere. Soil Biology and Biochemistry 30, 36–78. HAGN, A., PRITSCH, K., SCHLOTER, M., MUNCH, J.C., 2003: Fungal diversity in agricultural soil under different farming management systems, with special reference to biocontrol strains of Trichoderma spp. Biology and Fertility of Soils 38, 236–244. HARMAN, G., HOWELL, C. R., VITERBO, A., CHET, I., LORITO, M., 2004a: Trichoderma spe- cies- opportunistic, avirulent plant symbionts. Nature Reviews 2, 43–56. HARMAN, G., LORITO, M., LYNCH, J. M., 2004b: Uses of Trichoderma spp. to alleviate or remediate soil and water pollution. Advances in Applied Microbiology 56, 313–330. HAWKSWORTH, D. L., ROSSMAN, A. Y., 1997: Where are all the undescribed fungi? Phyto- pathology 87, 888–891. HJELJORD, L., TRONSMO, A., 1998: Trichoderma and Gliocladium in biological control: an overview. In: HARMAN, G. E., KUBICEK, C. P. (eds.), Trichoderma and Gliocladium, 2. Enzymes, biological control and commercial applications, 131–151. Taylor and Francis Ltd., London. HOWELL, C. R., 1998: The role of antibiosis in Biocontrol. In: HARMAN, G. E., KUBICEK, C. P. (eds.), Trichoderma and Gliocladium, 2. Enzymes, biological control and commercial applications, 173–184. Taylor and Francis, London. 48 ACTA BOT. CROAT. 73 (1), 2014 MUNIAPPAN V., MUTHUKUMAR T. 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:57 Color profile: Generic CMYK printer profile Composite Default screen IBEKWE, A. M., KENNEDY, A. C., FROHNE, P. S., PAPRIERNIK, S. K., YANG, C. H., 2002: Mi- crobial diversity along a transect of agronomic zones. FEMS Microbiology Ecology 39, 183–191. JACKSON, M. L., 1971: Soil chemical analysis. Prentice Hall, New Delhi, India. KREDICS, L., ANTAL, Z., MANCZINGER, L., SZEKERES, A., KEVEI, F., NAGY, E., 2003: Influ- ence of environmental parameters on Trichoderma strains with biocontrol potential. Food Technology and Biotechnology 47, 37–42. KÜÇÜK, C., KIVANÇ, M., 2003: Isolation of Trichoderma spp. and determination of their antifungal, biochemical and physiological features. Turkish Journal of Biology 27, 247–253. KUMAR, R., MAURYA, S., KUMARI, A., CHOUDHARY, J., DAS, B., NAIK, S. K., KUMAR, S., 2012: Biocontrol potentials of Trichoderma harzianum against sclerotial fungi. Bioscan 7: 521–525. LEWIS, J. A., PAPAVIZAS, G. C., 1991: Biocontrol of plant diseases: the approach of tomor- row. Crop Protection 10, 95–105. MANJULA, K., KRISHNA KISHORE, G., GIRISH, A. G., SINGH, S. D., 2004: Combined applica- tion of Pseudomonas fluorescens and Trichoderma viride has an improved biocontrol activity against stem rot in groundnut. Plant Pathology Journal 20: 75–80 MARTIN, J. P., 1950: Use of acid rose Bengal and Streptomycin in the plate method for esti- mating soil fungi. Soil Science 69, 215–232. NELSON, P. E., TOUSSOUN, T. A., MARASAS, W. F. O., 1983: Fusarium species: an illustrated manual for identification. The Pennsylvania State Press, London. OKOTH, S., OKOTH, P., MUYA, E., 2009: Influence of soil chemical and physical properties on occurrence of Trichoderma spp. in Embu, Kenya. Tropical and Subtropical Agroeco- systems 11, 303–312. PAPAVIZAS, G. C., 1985: Trichoderma and Gliocladium biology, ecology and potential for biocontrol. Annual Review of Phytopathology 23, 23–54. PITT, I. J., 1979: The Genus Penicillium and its telomorphic states Eupenecillium and Tala- romyces. Academic Press, New York. RAO, M. R., ONG, C. K., PATHAK, P., SHARMA, M. M., 1991: Productivity of annual cropping and agroforestry systems on a shallow Alfisols in semi-arid India. Agroforestry Systems 15, 51–63. RIFAI, M. A., 1969: A revision of the genus Trichoderma. Mycological Papers 116, 1–56. RUST, R. H., 1983: Alfisols. In: WILDING, L. P., SMECK, N. E., HALL, G. F. (eds.), Pedoge- nesis and soil taxonomy: II. The soil orders, 253–282. Elsevier, New York. SAMUELS, G. J., 2006: Trichoderma: systematics, the sexual state and ecology. Phytopath- ology 96, 195–206. SAMUELS, G. J., PARDO-SCHULTHEISS, R., HEBBAR, P., LUMSDEN, R. D., BASTOS, C. N., COSTA, J. C., BEZERRA, J. L., 2000: Trichoderma stromaticum sp. nov., a parasite of the cocoa witches broom pathogen. Mycological Research 104, 760–764. SARIAH, M., CHOO, C. W., ZAKARIA, H., NORIHAN, M. S., 2005: Quantification and characte- rization of Trichoderma spp. from different ecosystems. Mycopathology 159, 113–117. ACTA BOT. CROAT. 73 (1), 2014 49 INFLUENCE OF CROP SPECIES AND EDAPHIC FACTORS ON TRICHODERMA 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:57 Color profile: Generic CMYK printer profile Composite Default screen SIMON, A., SIVASITHAMBARAM, K., MACNISH, G. C., 1988: Effect of application of soil nitro- genous fertilizers and lime on biological suppression of Gaeumannomyces graminis var. tritici. Transactions of the British Mycological Society 91, 287–294. SOIL SURVEY STAFF, 1994: Key to soil taxonomy. Sixth Edition, Pocahontas Press., Inc., Blacksburg, Virginia. SUBRAMAINAN, C. V., 1970: Hypomycetes. ICARI, New Delhi. WAKELIN, S. A., SIVASITHAMPARAM, K., COLE, A. L. J., SKIPP, R. A., 1999: Saprophytic growth in soil of a strain of Trichoderma koningii. New Zealand Journal of Agricultural Research 42, 337–345. WEBSTER, J., RIFAI, M. A., 1968: Culture studies on Hypocrea and Trichoderma IV. Hypo- crea piluifera sp.nov. Transactions of the British Mycological Society 51, 511–514. WEBSTER, J., 1964: Culture studies on Hypocrea and Trichoderma I. comparison of perfect and imperfect states of H. gelatinosa, H. rufa and Hypocrea sp. Transactions of the Brit- ish Mycological Society 47, 75–96. WELLS, H. D., 2000: Trichoderma as a biochemical agent. In: MUKERJI, K. G., GARG, K. L. (eds.), Biocontrol of plant diseases, 1, 71–82. CRC Press Inc., Boca Raton, Florida. WHIPPS, J. M., 2001: Microbial interactions and biocontrol in the rhizosphere. Journal of Experimental Botany 52, 487–511. WIDDEN, P., 1984: The effects of temperature on competition for spruce needles among sympatric species of Trichoderma. Mycologia 76, 873–883. WIDDEN, P., 1987: Fungal communities in soils along an elevation gradient in northern Eng- land. Mycologia 79, 298–309. 50 ACTA BOT. CROAT. 73 (1), 2014 MUNIAPPAN V., MUTHUKUMAR T. 741 Muniappan and Muthukumar.ps U:\ACTA BOTANICA\Acta-Botan 1-14\741 Muniappan and Muthukumar.vp 19. o ujak 2014 16:46:57 Color profile: Generic CMYK printer profile Composite Default screen