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Biological control of one species belonging to the dominant mycobiota of rice of Valencia

Biological control of one species belonging to the dominant mycobiota of rice of Valencia Annals of Microbiology, 58 (1) 7-14 (2008) Biological control of one species belonging to the dominant mycobiota of rice of Valencia Francisca SEMPERE, María Pilar SANTAMARINA Department of Agroforest Ecosystems. School of Rural Environments and Enology; Polytechnic University of Valencia. Avda. Blasco Ibañez 21, 46010 Valencia, Spain Received 28 June 2007 / Accepted 28 December 2007 Abstract - The possible biological control of the seed-associated fungus, Nigrospora oryzae by Trichoderma harzianum under differ- ent environmental conditions was investigated. A study of the fungal growth in dual cultures revealed that T. harzianum inhibited by contact N. oryzae at all testing temperatures and water activities tested except at 0.95 a and 15 ºC, where T. harzianum inhibited pathogen growth before hyphal contact and exhibited an inhibition zone between the colonies of both fungi. Suppression of the sporu- lation, loss of turgor and cell collapse, wall’s disintegration, coiling and penetration of T. harzianum around different structures of N. oryzae were observed by cryo-scanning electron microscopy. The effect of abiotic factors water activity and temperature on fungal growth was determined. Key words: mycoparasitism, Nigrospora oryzae, Index of Dominance, rice, Trichoderma harzianum, water activity. INTRODUCTION (Hjeljord and Tronsmo, 1998; Hermosa et al., 2000). Others fungi have been studied with this purpose. In this Rice (Oryza sativa L.) is one of the most important cereal way, Penicillium oxalicum and species de Trichoderma and grains grown worldwide. This crop is a primary food source Gliocadium have been successfully applied controlling rice for more than a third of the world’s population and provides pathogens Rhizoctonia solani, Alternaria alternata, 27% of the dietary energy supply, and 20% of the dietary Nigrospora oryzae and Bipolaris oryzae (Rosales and Mew, protein intake (Kush, 1997; Fresco, 2005). 1982; Roy and Sayre, 1984; Gokulapalan and Nair, 1986; Fungal diseases are one of the major factors limiting Abdel-Fattah et al., 2007; Sempere and Santamarina, rice production (Kim et al., 2003). Biological control of fun- 2007a, 2007b). gal plant pathogens appears as an attractive and realistic In the Valencian autonomous region, typical rice pro- approach, and numerous microorganisms have been iden- duction area, which owns the origin denomination Rice of tified as biocontrol agents (Massart and Jijakli, 2006). Valencia, Pyricularia, Alternaria, Phoma, Bipolaris, Studies made for suppressing these cereal pathogens are Nigrospora, Fusarium, Aspergillus, and Penicillium genera few. Within these studies Pseudomonas fluorescens, Delftia have been isolated. Among them the seedborne fungus tsuruhatensis, Bacillus mycoides and Streptomyces spp. Nigrospora oryzae (Berk. & Broome) Petch (teleomorph: bacteria have been used as antagonistic against Khushia oryzae Huds.) has been found with high percentage Sarocladium oryzae, Rhizoctonia solani, Drechslera oryzae (Piñeiro and García, 2000; Santamarina et al., 2002). This and Pyricularia oryzae (Sakthivel and Gnanamanickam, species affects different parts of the rice plant but stands out because of its contribution for pecky rice. Infected grains 1986a, 1986b; Sakthivel et al., 1986; Chakrabarti and Chaudhari, 1992; Krishnamurthy and Gnanamanickam, are discoloured and sometimes black and white coloured 1998; Tian et al., 2004; Han et al., 2005; Nagarajkumar et due to the presence of mycelial mass and contamination can al., 2004, 2005). Candida sp. and Sporobolomyces roseus occur preharvest, harvest and rice processing. yeasts have been employed too against these strains (Akai Understanding how biocontrol agents exert their pro- and Kuramoto, 1968; Rush et al., 1998). tective effects is a prerequisite to their effective practical Trichoderma species have been investigated as biologi- application (Massart and Jikali, 2006). The purpose of this paper was to investigate the interaction between T. cal control agents (BCAs) for over 70 years, but it is only recently that strains have become commercially available harzianum and N. oryzae in co-culture at different environ- mental conditions and culture media using different tech- niques. In addition, an ecophysiological study of both * Corresponding author. Phone: +34 963 877414; strains was realised. E-mail: frasemfe@yahoo.es 8 F. Sempere and M.P. Santamarina MATERIALS AND METHODS species were inoculated in REA squares 5 mm apart, mounted on a glass slide in a glass rod inside a Petri plate Fungal strains. Nigrospora oryzae DAE 7406 was isolated of 90 mm under conditions of total asepsis. To maintain the from rice grains collected from different fields and cooper- levels of water activity, filter paper disks impregnated with atives of the main rice producing areas in Valencia. different water activity levels (0.995, 0.98 and 0.95 Trichoderma harzianum CECT 20736 was isolated from obtained with 2.5, 11 and 23.5 g glycerol/100 ml distilled national corn grain samples. water) were aseptically placed on the Petri plates. For the The above mentioned strains were kept in the analysis of fungal interaction in rice grains, first the sam- Department of Agroforest Ecosystems of the School of ples were sterilised with a solution of sodium hypochlorite. Rural Environments and Enology, Polytechnic University of For setting the water activities, the rice grains were Valencia, Spain. deposited 48 h in the solutions described above. Water activity was measured and set with an Aqualab (Decagon, Growth in paired cultures. The basic medium used was Inc., Pullman, WA, USA). The rice grains were deposited on Rice Extract Agar (REA) with a pH of 5.5. The water activ- a glass slide as described above. The fungal species were ity (a ) of this basal medium was 0.995, and this a was inoculated every 3 mm (Sempere and Santamarina, 2006b, w w modified by the addition of different amounts of glycerol to 2007a). obtain a levels of 0.98, 0.95, 0.90 and 0.85 (Sempere and Microscopic examination of the dual microculture was Santamarina, 2006a). The experiments were carried out at performed between 5-30 days after inoculation. 15 and 25 ºC in the dark. The fungi disks of T. harzianum and N. oryzae (8 mm Assessment of antibiosis. Growth of N. oryzae jointly T. diameter) obtained from the growing margins of the fungus harzianum was measured. Treatments were compared with colonies grown in Potato Dextrose Agar (PDA) at 25 ºC for the diameter of each REA plate control inoculated with N. 5 days, were inoculated at opposite sides -45 mm apart-, oryzae. of a 90 and 150 mm Petri plates containing REA. Plates with the same water activities were placed in Statistical analysis. The analysis of variance (ANOVA) water impermeable plastic containers together with two with significance values of P < 0.01 was used to determine 100 ml beakers containing a glycerol water solution with an the influence of parameters water activity and temperature equilibrium relative humidity value identical to the a of the (T), and of their interaction (a x T) on dual fungal growth w w plates. In this way, equilibration to the target a levels was rates. STATGRAPHICS Plus 5.0 software (Stat Point, Inc., achieved within 24 h, maintaining a constant relative Herndon, Virginia, USA) was used in the study. humidity inside the Petri dishes and also controlling the a of the substrate. Fungal growth was measured at right angles, five days RESULTS AND DISCUSSION at intervals of 24 h after inoculation. A linear regression of the data was performed in order to calculate the growth Effects of water activity and temperature on mycelial -1 rate (mm·day ). The computer software used was growth Microsoft Excel 2003. Figure 1 shows growth rates of T. harzianum and N. oryzae The experiment was repeated four times. dually grown at 15, 25 ºC and at five different water activ- ities. Macroscopic experiments. Macroscopic experiments Growth of the species studied was fastest at the high- were conducted in 60 days. Growth rates were obtained est a studied at all temperatures. The optimum a (0.995) w w and Petri plates at 0.95, 0.98 and 0.995 water activities for growth of both strains didn’t vary with temperature. The and both temperatures were examined macroscopically, maximum growth rates occurred at 25 ºC and 0.995 a for the type of interactions was determined, and numerical T. harzianum and N. oryzae. The growth rates of T. -1 scores were assigned to obtain an Index of Dominance harzianum and N. oryzae were of 16 mm day and 8.97 -1 according to method proposed by Magan and Lacey (1984). mm day respectively. Mutual intermingling (1); mutual antagonism on contact or At 25 ºC and five days, the minimum a for growth was with free space between fungus colonies < 2 mm (2); 0.90 for N. oryzae and 0.95 for T. harzianum. At 15 ºC the mutual antagonism at a distance (3); dominance on con- tact (4 for the dominant species, 0 for the inhibited species); dominance at a distance (5 for the dominant species, 0 for the inhibited species). TABLE 1 - Analysis of variance of the growth rate of Trichoderma Analysis of Petri plates in this experiment for water harzianum and Nigrospora oryzae; significance of activities of 0.90 and 0.85 was discarded. water activity (a ), temperature (T) and their interac- tion (a x T) Microscopic experiments. For cryo-scanning electron Factor DF MS F-ratio P-value microscopy (cryo-SEM), two experiments were undertaken. Experiment 1: both strains were inoculated at the same a 4 5826.55 97.40 0.0000** time. Experiment 2: N. oryzae was inoculated three days T 1 4695.68 78.50 0.0000** later inoculation of T. harzianum. Microscopic analysis was performed on Rice Extract Agar and full rice grain. The rice a x T 4 1196.92 20.01 0.0000** grain variety used in the experiment was Bomba, typical of the designation of origin Rice of Valencia. DF: degrees of freedom, MS: mean squares. ** Indicates that the factor elicited a significant effect (P < 0.01). To study hyphal interactions in Rice Extract Agar, both Ann. Microbiol., 58 (1), 7-14 (2008) 9 minimum a was 0.95 for N. oryzae and 0.98 for T. Macroscopic observations of dual culture harzianum. Growth of both species at 15 ºC and 25 ºC was Regarding the Index of Dominance (Table 2) and the Fig. minimal at 0.90 a in REA during the eight testing weeks. 2, dominance on contact was the most common interaction Trichoderma harzianum and N. oryzae did not show any of T. harzianum against N. oryzae. Trichoderma harzianum growth at 0.85 a . was assigned a value of 4 and N. oryzae a value of 0. Similar results were obtained previously when N. oryzae In Petri plates dual cultures, the first apparent contact and T. harzianum grown individually and jointly Alternaria between hyphae of the two fungi occurred at 0.995 a 25 alternata (Sempere and Santamarina, 2006b, 2007a). ºC, 4 days after inoculation. As long as the water activity Trichoderma harzianum presented higher growth rates and temperature decreased the colonies got in contact than N. oryzae at both temperatures, for high water activ- later. In the subsequent days, T. harzianum mycelium con- ities studied, 0.995 and 0.98. At 0.95 a the growth of N. tinued growing and colonising the substratum already oryzae was bigger (Fig. 1). colonised by N. oryzae. The aerial mycelium and sporula- tion of T. harzianum were more abundant when this strain grew over N. oryzae. 15°C In the Fig. 2 it can be seen the colonies of T. harzianum, 6.57 T. harzianum 6 which are greenish and whitish in colour grew over the N. oryzae colonies of N. oryzae. The colonies of N. oryzae grown alone were first brown with a wool-like aerial white myceli- 3.77 3.8 um irregularly distributed, more abundant at 25 than at 15 ºC. On aging, their colour changed to darker brown-black by the growing margin of the fungus colony and to brown 1.46 in the middle (Sempere and Santamarina, 2006b). When 0.62 this species faced to T. harzianum in some water activities 0 0 these mycelial features were not observed even the N. 0.85 0.90 0.95 0.98 0.995 oryzae colony was not detected. In this way this assay observed that one of the mecha- nisms that presented T. harzianum as a biocontrol agent at 25 °C high water activities, apart from temperature, was the T. harzianum 16 16 competition for space and nutrients. This mechanism was N. oryzae 14 described to play a role in the biocontrol of Verticillium 12 dahliae and Rhizoctonia solani by this strain (Santamarina 10 and Roselló, 2006). 9.5 8.97 8 In dual culture of T. harzianum and N. oryzae at 0.95 a and 15 ºC, an inhibition zone around the Nigrospora colony 5.65 2.97 was observed, where macroscopically, no apparent contact of the hyphae of the two fungi was observed (Fig. 2). 0.3 1.49 According to method proposed by Magan and Lacey 0.85 0.90 0.95 0.98 0.995 (1984), mutual antagonism at a distance happens when a separation between both fungal species of more than 2 mm is observed. A value of 3 is assigned for each fungal strain. FIG. 1 - Ecophysiological study of Trichoderma harzianum and The authors have found that this antagonism is not mutu- Nigrospora oryzae dual culture in Rice Extract Agar at al. Trichoderma harzianum colony antagonised N. oryzae. different temperatures and water activities (a ). Thus when comparing the growth rates of N. oryzae grown individually with the results obtained from the strain grown Expansion of mycelia of both colonies was the same in jointly T. harzianum, second decreased. Nevertheless the the direction of the other colony than it was when they were grown individually. When the colonies got in contact, N. oryzae growth rate decreased in particular at the fungal TABLE 2 - Index of Dominance (I ) at different temperatures interaction front line. However at 0.95 a and 15 ºC T. and water activities harzianum inhibited pathogen growth before hyphal con- tact and exhibited an inhibition zone between the colonies Temp. Fungus a I w D of both fungi (Fig. 2). Thangavelu et al. (2004) found that species 0.995 0.98 0.95 0.90 Trichoderma harzianum was the most effective in inhibiting 25 ºC T. harzianum 444 X 12 the mycelial growth of Fusarium oxysporum f. sp. cubense N. oryzae 000 X 0 in vitro. Growth of F. oxysporum f. sp. ciceris was also sup- pressed by T. harzianum when these species were co-cul- 15 ºC T. harzianum 443 X 11 tured on PDA (Hervás et al., 1998, Dubey et al., 2007). N. oyzae 003 X 3 Biotic and abiotic parameters determine the extent of fungal colonisation and, among them, water activity, tem- I refers to sum of scores at 25 and 15 ºC for Trichoderma perature and fungal interactions are the most important harzianum competing with Nigrospora oryzae based on the (Torres et al., 2003). In this study, significant differences interaction scores for each species. Dominance on contact (4 for in water activity and temperature effects on growth rates the dominant species T. harzianum, 0 for the inhibited species were registered for T. harzianum and N. oryzae (P < 0.001) N. oryzae). Mutual antagonism at a distance (3 for both species). (X) Analysis of interaction was discarded. (Table 1). -1 -1 Growth rate (mm day ) Growth rate (mm day ) 10 F. Sempere and M.P. Santamarina 0.95 a 0.98 a 0.995 a w w w 0.95 a 0.98 a 0.995 a w w w FIG. 2 - Petri plates showing the interaction between Trichoderma harzianum (left) and Nigrospora oryzae (right) after 8 weeks at dif- ferent water activities and temperatures. Row A: 25 ºC. Culture of N. oryzae was inhibited by T. harzianum. Both species were growing until both colonies got in contact. Later T. harzianum grew trough N. oryzae. Row B: 15 ºC. The same type of interaction of Row A was registered (0.98 and 0.995 a ), while T. harzianum inhibited N. oryzae growth at a distance (0.95 a ), colony of T. harzianum. growth of T. harzianum was very similar in both situations Microscopic observation of the inoculation of N. oryzae (data not shown). three days later inoculation of T. harzianum revealed the Finally, the numerical values used in the Index of mechanism of mycoparasitism at all conditions tested Dominance revealed that T. harzianum was the dominant except at 0.95 a and 15 ºC. One of the earliest events of species over N. oryzae at 25 and 15 ºC (Table 2). the antagonistic process was the apparent affinity of T. harzianum for hypha and conidia of the N. oryzae (Fig. 3C Mycelial interactions between Trichoderma harzia- and 3D). However, how Trichoderma species “recognise” num and Nigrospora oryzae their host and attack host cells is yet unknown. This When T. harzianum and N. oryzae were inoculated at the process may involve hydrophobic interactions or interac- same time, conidia of N. oryzae were not observed (data tions between complementary molecules present on the not shown). Macroscopic examination of the Petri plates surface of both the host and the mycoparasite such as showed discoloured aerial mycelium of N. oryzae (Fig. 2). between lectins and carbohydrates (Whipps, 2001). These observations suggested that T. harzianum sup- Different researchers worked in this mycoparasite and oth- pressed sporulation of its host. ers species for elucidated this process and signalling path- Previously, this capacity was proposed as a potential ways that occur latter recognition of the host (Elad et al., strategy of biological control (Sutton and Peng, 1993; Köhl 1983; Inbar and Chet, 1994; Neethling and Nevalainen, and Fokkema, 1998). In different studies, the antagonist 1996; Omero et al., 1999). Clonostachys rosea reduced and suppressed conidia of Occasionally T. harzianum hyphae coiled around the Botrytis cinerea (Sutton et al., 1997; Morandi et al., 2003). hyphae and conidia of the pathogen from where penetra- This behaviour was also observed when mycoparasitic tion took place (Fig. 3E and 4D). Also, directly penetration Pythium oligandrum was confronted against Fusarium cul- of T. harzianum into hyphae and conidia of N. oryzae was morum in dual culture (Davanlou et al., 1999). Studies of observed (Fig. 3F, 4A, 4C, and 4D). Microscopic examina- dual cultures of T. harzianum and other species revealed tions showed loss of turgor and cell collapse of N. oryzae inhibition of germination but no suppression of conidia conidium (Fig. 3B) and disintegrated hyphal walls. Finally, (Lorito et al., 1993a, 1993b; Zimand et al., 1996; Kapat et T. harzianum utilised both their cell walls and cellular con- al., 1998; Golam Mortuza and Ilag, 1999). tents for nutrition and come out producing sexual struc- Individual culture of N. oryzae sporulated at all condi- tures (Fig. 3F, 4A, 4C, and 4D). tions tested (0.995, 0.98 and 0.95 a at 15 and 25 ºC). For The enzymes chitinases and glucanases from other strain belonging to the same species isolated from Trichoderma harzianum have been demonstrated to play the rice grains of Valencia, only formed conidia at 0.995 an important role in mycoparasitic action against different and 0.98 a at 25 ºC (Sempere and Santamarina, 2006b). fungi and others organisms (Viterbo et al., 2002; Ezziyyani This filamentous fungus formed unicellular solitary conidia, et al., 2004; Limón et al., 2004; Binod et al., 2007). globose or subglobose shaped with a smooth surface tex- However, fungal proteases may be significantly involved in ture, irregularly grown from short or ill-defined conidio- antagonistic activity, not only in the breakdown of the host phores (Fig. 3A and 3B). cell walls but also by inactivation of enzymes of pathogens Ann. Microbiol., 58 (1), 7-14 (2008) 11 A B 0.995 a , 15 ºC, X2000 0.995 a , 25 ºC, X3500 C D 0.98 a , 15 ºC, X2000 0.95 a , 25 ºC, X5000 w w D E 0.98 a , 25 ºC, X2000 0.995 a , 25 ºC ,X3500 w w FIG. 3 - Mycoparasitic interactions between Trichoderma harzianum and Nigrospora oryzae. A and B: conidia and conidiophores of N. oryzae, it can be seen their morphology still undamaged; C and D: conidia and hypha of N. oryzae in the beginning of being attacked by T. harzianum; E: hypha of T. harzianum coiling around hypha of N. oryzae (arrows); F: direct penetration of T. harzianum into partially disintegrated conidia of N. oryzae. A B 0.995 a , 15 ºC, X1000 0.98 a , 25 ºC, X7500 w w C D 0.95 a , 25 ºC, X2000 0.995 a , 25 ºC, X2000 w w FIG. 4 - Mycoparasitic interactions between Trichoderma harzianum and Nigrospora oryzae. A: directly penetration of T. harzianum into partially disintegrated conidia of N. oryzae (arrows); B: detail of the figure A, loss of turgor and cell collapse of N. oryzae conidium caused by T. harzianum; C: conidia of T. harzianum coming out of a degraded hypha of N. oryzae (arrow); D: hypha of T. harzianum coiling around, penetrating and coming out a conidium of N. oryzae. 12 F. Sempere and M.P. Santamarina nd (Rodriguez-Kabana et al., 1978, Suarez et al., 2004). Diseases of Cereals and Pulses, 2 edn., Prentice-Hall Inc., USA, pp. 116-129. Proteolytic enzymes of T. harzianum reduced Botrytis cinerea germination and deactivated partially hydrolytic Cook R.J. (1993). Making greater use of introduced microorgan- isms for biological control of plant pathogens. Annu. Rev. enzymes endo-PG and exo-PG produced by this strain (Elad Phytopathol., 31: 53-80. and Kapat, 1999). Cryo-SEM study in the interaction zone, Davanlou M., Madsen A.M., Madsen C.H., Hockenhull J. 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There Fourteenth Annual Meeting of the Mycological Society of are few researches and the usual techniques to study fun- India and Seminar on Applied Micology, Thanjavur, India, p. gal relations are tedious SEM preparations or light micro- scopical observations (Madi et al., 1997; Davanlou et al., Golam Mortuza M., Ilag L.L. (1999). Potential for biocontrol of 1999; Moussa, 2002; Zadworny et al., 2004). Lasiodiplodia theobromae (Pat.) Griff. & Maubl. in banana fruits by Trichoderma species. Biol. Control, 15: 235-240. Knowledge of the mechanism of action involved in the biocontrol process can permit establishment of optimum Gupta V.P., Tewari S.K., Govindaiah, Bajpai A.K. (1999). Ultrastructure of mycoparasitism of Trichoderma, conditions for the interaction between the pathogen and Gliocladium and Laetisaria species on Botryodiplodia theo- the biological control agent and is important for imple- bromae. J. 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La sanidad en el arrozal valenciano. Zhou S.N. (2004). Study on the communities of endophytic Enfermedades. Vida Rural, 108: 26-30. fungi and endophytic actinomycetes from rice and their Punja Z.K., Utkhede R.S. (2003). Using fungi and yeasts to man- antipathogenic activities in vitro. World J. Microbiol. age vegetable crop diseases. Trends Biotechnol., 21 (9): Biotechnol., 20: 303-309. 400-407. Torres M.R., Ramos A.J., Soler J., Sanchis V., Marín S. (2003). Rodriguez-Kabana R., Kelley W.D., Curl E.A. (1978). Proteolytic SEM study of water activity and temperature effects on the activity of Trichoderma viride in mixed culture with 14 F. Sempere and M.P. Santamarina initial growth of Aspergillus ochraceus, Alternaria alternata Zadworny M., Werner A., Idzikowska K. (2004). Behaviour of the and Fusarium verticillioides on maize grain. Int. J. Food hyphae of Laccaria laccata in the presence of Trichoderma Microbiol., 81: 185-193. harzianum in vitro. Mycorrhiza, 14: 401-405. Viterbo A., Montero M., Ramot O., Friesem D., Monte E., Llobell Zimand G., Elad Y., Chet I. (1996) Effect of Trichoderma A., Chet I. (2002). Expression regulation of the endochiti- harzianum on Botrytis cinerea pathogenicity. nase chit36 from Trichoderma asperellum (T. harzianum T- Phytopathology, 86: 1255-1260. 203). Curr. Genet., 42: 114-122. Whipps J.M. (2001). Microbial interactions and biocontrol in the rhizosphere. J. Exp. Bot., 52: 487-511. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Biological control of one species belonging to the dominant mycobiota of rice of Valencia

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Springer Journals
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Copyright © 2008 by University of Milan and Springer
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/BF03179438
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Abstract

Annals of Microbiology, 58 (1) 7-14 (2008) Biological control of one species belonging to the dominant mycobiota of rice of Valencia Francisca SEMPERE, María Pilar SANTAMARINA Department of Agroforest Ecosystems. School of Rural Environments and Enology; Polytechnic University of Valencia. Avda. Blasco Ibañez 21, 46010 Valencia, Spain Received 28 June 2007 / Accepted 28 December 2007 Abstract - The possible biological control of the seed-associated fungus, Nigrospora oryzae by Trichoderma harzianum under differ- ent environmental conditions was investigated. A study of the fungal growth in dual cultures revealed that T. harzianum inhibited by contact N. oryzae at all testing temperatures and water activities tested except at 0.95 a and 15 ºC, where T. harzianum inhibited pathogen growth before hyphal contact and exhibited an inhibition zone between the colonies of both fungi. Suppression of the sporu- lation, loss of turgor and cell collapse, wall’s disintegration, coiling and penetration of T. harzianum around different structures of N. oryzae were observed by cryo-scanning electron microscopy. The effect of abiotic factors water activity and temperature on fungal growth was determined. Key words: mycoparasitism, Nigrospora oryzae, Index of Dominance, rice, Trichoderma harzianum, water activity. INTRODUCTION (Hjeljord and Tronsmo, 1998; Hermosa et al., 2000). Others fungi have been studied with this purpose. In this Rice (Oryza sativa L.) is one of the most important cereal way, Penicillium oxalicum and species de Trichoderma and grains grown worldwide. This crop is a primary food source Gliocadium have been successfully applied controlling rice for more than a third of the world’s population and provides pathogens Rhizoctonia solani, Alternaria alternata, 27% of the dietary energy supply, and 20% of the dietary Nigrospora oryzae and Bipolaris oryzae (Rosales and Mew, protein intake (Kush, 1997; Fresco, 2005). 1982; Roy and Sayre, 1984; Gokulapalan and Nair, 1986; Fungal diseases are one of the major factors limiting Abdel-Fattah et al., 2007; Sempere and Santamarina, rice production (Kim et al., 2003). Biological control of fun- 2007a, 2007b). gal plant pathogens appears as an attractive and realistic In the Valencian autonomous region, typical rice pro- approach, and numerous microorganisms have been iden- duction area, which owns the origin denomination Rice of tified as biocontrol agents (Massart and Jijakli, 2006). Valencia, Pyricularia, Alternaria, Phoma, Bipolaris, Studies made for suppressing these cereal pathogens are Nigrospora, Fusarium, Aspergillus, and Penicillium genera few. Within these studies Pseudomonas fluorescens, Delftia have been isolated. Among them the seedborne fungus tsuruhatensis, Bacillus mycoides and Streptomyces spp. Nigrospora oryzae (Berk. & Broome) Petch (teleomorph: bacteria have been used as antagonistic against Khushia oryzae Huds.) has been found with high percentage Sarocladium oryzae, Rhizoctonia solani, Drechslera oryzae (Piñeiro and García, 2000; Santamarina et al., 2002). This and Pyricularia oryzae (Sakthivel and Gnanamanickam, species affects different parts of the rice plant but stands out because of its contribution for pecky rice. Infected grains 1986a, 1986b; Sakthivel et al., 1986; Chakrabarti and Chaudhari, 1992; Krishnamurthy and Gnanamanickam, are discoloured and sometimes black and white coloured 1998; Tian et al., 2004; Han et al., 2005; Nagarajkumar et due to the presence of mycelial mass and contamination can al., 2004, 2005). Candida sp. and Sporobolomyces roseus occur preharvest, harvest and rice processing. yeasts have been employed too against these strains (Akai Understanding how biocontrol agents exert their pro- and Kuramoto, 1968; Rush et al., 1998). tective effects is a prerequisite to their effective practical Trichoderma species have been investigated as biologi- application (Massart and Jikali, 2006). The purpose of this paper was to investigate the interaction between T. cal control agents (BCAs) for over 70 years, but it is only recently that strains have become commercially available harzianum and N. oryzae in co-culture at different environ- mental conditions and culture media using different tech- niques. In addition, an ecophysiological study of both * Corresponding author. Phone: +34 963 877414; strains was realised. E-mail: frasemfe@yahoo.es 8 F. Sempere and M.P. Santamarina MATERIALS AND METHODS species were inoculated in REA squares 5 mm apart, mounted on a glass slide in a glass rod inside a Petri plate Fungal strains. Nigrospora oryzae DAE 7406 was isolated of 90 mm under conditions of total asepsis. To maintain the from rice grains collected from different fields and cooper- levels of water activity, filter paper disks impregnated with atives of the main rice producing areas in Valencia. different water activity levels (0.995, 0.98 and 0.95 Trichoderma harzianum CECT 20736 was isolated from obtained with 2.5, 11 and 23.5 g glycerol/100 ml distilled national corn grain samples. water) were aseptically placed on the Petri plates. For the The above mentioned strains were kept in the analysis of fungal interaction in rice grains, first the sam- Department of Agroforest Ecosystems of the School of ples were sterilised with a solution of sodium hypochlorite. Rural Environments and Enology, Polytechnic University of For setting the water activities, the rice grains were Valencia, Spain. deposited 48 h in the solutions described above. Water activity was measured and set with an Aqualab (Decagon, Growth in paired cultures. The basic medium used was Inc., Pullman, WA, USA). The rice grains were deposited on Rice Extract Agar (REA) with a pH of 5.5. The water activ- a glass slide as described above. The fungal species were ity (a ) of this basal medium was 0.995, and this a was inoculated every 3 mm (Sempere and Santamarina, 2006b, w w modified by the addition of different amounts of glycerol to 2007a). obtain a levels of 0.98, 0.95, 0.90 and 0.85 (Sempere and Microscopic examination of the dual microculture was Santamarina, 2006a). The experiments were carried out at performed between 5-30 days after inoculation. 15 and 25 ºC in the dark. The fungi disks of T. harzianum and N. oryzae (8 mm Assessment of antibiosis. Growth of N. oryzae jointly T. diameter) obtained from the growing margins of the fungus harzianum was measured. Treatments were compared with colonies grown in Potato Dextrose Agar (PDA) at 25 ºC for the diameter of each REA plate control inoculated with N. 5 days, were inoculated at opposite sides -45 mm apart-, oryzae. of a 90 and 150 mm Petri plates containing REA. Plates with the same water activities were placed in Statistical analysis. The analysis of variance (ANOVA) water impermeable plastic containers together with two with significance values of P < 0.01 was used to determine 100 ml beakers containing a glycerol water solution with an the influence of parameters water activity and temperature equilibrium relative humidity value identical to the a of the (T), and of their interaction (a x T) on dual fungal growth w w plates. In this way, equilibration to the target a levels was rates. STATGRAPHICS Plus 5.0 software (Stat Point, Inc., achieved within 24 h, maintaining a constant relative Herndon, Virginia, USA) was used in the study. humidity inside the Petri dishes and also controlling the a of the substrate. Fungal growth was measured at right angles, five days RESULTS AND DISCUSSION at intervals of 24 h after inoculation. A linear regression of the data was performed in order to calculate the growth Effects of water activity and temperature on mycelial -1 rate (mm·day ). The computer software used was growth Microsoft Excel 2003. Figure 1 shows growth rates of T. harzianum and N. oryzae The experiment was repeated four times. dually grown at 15, 25 ºC and at five different water activ- ities. Macroscopic experiments. Macroscopic experiments Growth of the species studied was fastest at the high- were conducted in 60 days. Growth rates were obtained est a studied at all temperatures. The optimum a (0.995) w w and Petri plates at 0.95, 0.98 and 0.995 water activities for growth of both strains didn’t vary with temperature. The and both temperatures were examined macroscopically, maximum growth rates occurred at 25 ºC and 0.995 a for the type of interactions was determined, and numerical T. harzianum and N. oryzae. The growth rates of T. -1 scores were assigned to obtain an Index of Dominance harzianum and N. oryzae were of 16 mm day and 8.97 -1 according to method proposed by Magan and Lacey (1984). mm day respectively. Mutual intermingling (1); mutual antagonism on contact or At 25 ºC and five days, the minimum a for growth was with free space between fungus colonies < 2 mm (2); 0.90 for N. oryzae and 0.95 for T. harzianum. At 15 ºC the mutual antagonism at a distance (3); dominance on con- tact (4 for the dominant species, 0 for the inhibited species); dominance at a distance (5 for the dominant species, 0 for the inhibited species). TABLE 1 - Analysis of variance of the growth rate of Trichoderma Analysis of Petri plates in this experiment for water harzianum and Nigrospora oryzae; significance of activities of 0.90 and 0.85 was discarded. water activity (a ), temperature (T) and their interac- tion (a x T) Microscopic experiments. For cryo-scanning electron Factor DF MS F-ratio P-value microscopy (cryo-SEM), two experiments were undertaken. Experiment 1: both strains were inoculated at the same a 4 5826.55 97.40 0.0000** time. Experiment 2: N. oryzae was inoculated three days T 1 4695.68 78.50 0.0000** later inoculation of T. harzianum. Microscopic analysis was performed on Rice Extract Agar and full rice grain. The rice a x T 4 1196.92 20.01 0.0000** grain variety used in the experiment was Bomba, typical of the designation of origin Rice of Valencia. DF: degrees of freedom, MS: mean squares. ** Indicates that the factor elicited a significant effect (P < 0.01). To study hyphal interactions in Rice Extract Agar, both Ann. Microbiol., 58 (1), 7-14 (2008) 9 minimum a was 0.95 for N. oryzae and 0.98 for T. Macroscopic observations of dual culture harzianum. Growth of both species at 15 ºC and 25 ºC was Regarding the Index of Dominance (Table 2) and the Fig. minimal at 0.90 a in REA during the eight testing weeks. 2, dominance on contact was the most common interaction Trichoderma harzianum and N. oryzae did not show any of T. harzianum against N. oryzae. Trichoderma harzianum growth at 0.85 a . was assigned a value of 4 and N. oryzae a value of 0. Similar results were obtained previously when N. oryzae In Petri plates dual cultures, the first apparent contact and T. harzianum grown individually and jointly Alternaria between hyphae of the two fungi occurred at 0.995 a 25 alternata (Sempere and Santamarina, 2006b, 2007a). ºC, 4 days after inoculation. As long as the water activity Trichoderma harzianum presented higher growth rates and temperature decreased the colonies got in contact than N. oryzae at both temperatures, for high water activ- later. In the subsequent days, T. harzianum mycelium con- ities studied, 0.995 and 0.98. At 0.95 a the growth of N. tinued growing and colonising the substratum already oryzae was bigger (Fig. 1). colonised by N. oryzae. The aerial mycelium and sporula- tion of T. harzianum were more abundant when this strain grew over N. oryzae. 15°C In the Fig. 2 it can be seen the colonies of T. harzianum, 6.57 T. harzianum 6 which are greenish and whitish in colour grew over the N. oryzae colonies of N. oryzae. The colonies of N. oryzae grown alone were first brown with a wool-like aerial white myceli- 3.77 3.8 um irregularly distributed, more abundant at 25 than at 15 ºC. On aging, their colour changed to darker brown-black by the growing margin of the fungus colony and to brown 1.46 in the middle (Sempere and Santamarina, 2006b). When 0.62 this species faced to T. harzianum in some water activities 0 0 these mycelial features were not observed even the N. 0.85 0.90 0.95 0.98 0.995 oryzae colony was not detected. In this way this assay observed that one of the mecha- nisms that presented T. harzianum as a biocontrol agent at 25 °C high water activities, apart from temperature, was the T. harzianum 16 16 competition for space and nutrients. This mechanism was N. oryzae 14 described to play a role in the biocontrol of Verticillium 12 dahliae and Rhizoctonia solani by this strain (Santamarina 10 and Roselló, 2006). 9.5 8.97 8 In dual culture of T. harzianum and N. oryzae at 0.95 a and 15 ºC, an inhibition zone around the Nigrospora colony 5.65 2.97 was observed, where macroscopically, no apparent contact of the hyphae of the two fungi was observed (Fig. 2). 0.3 1.49 According to method proposed by Magan and Lacey 0.85 0.90 0.95 0.98 0.995 (1984), mutual antagonism at a distance happens when a separation between both fungal species of more than 2 mm is observed. A value of 3 is assigned for each fungal strain. FIG. 1 - Ecophysiological study of Trichoderma harzianum and The authors have found that this antagonism is not mutu- Nigrospora oryzae dual culture in Rice Extract Agar at al. Trichoderma harzianum colony antagonised N. oryzae. different temperatures and water activities (a ). Thus when comparing the growth rates of N. oryzae grown individually with the results obtained from the strain grown Expansion of mycelia of both colonies was the same in jointly T. harzianum, second decreased. Nevertheless the the direction of the other colony than it was when they were grown individually. When the colonies got in contact, N. oryzae growth rate decreased in particular at the fungal TABLE 2 - Index of Dominance (I ) at different temperatures interaction front line. However at 0.95 a and 15 ºC T. and water activities harzianum inhibited pathogen growth before hyphal con- tact and exhibited an inhibition zone between the colonies Temp. Fungus a I w D of both fungi (Fig. 2). Thangavelu et al. (2004) found that species 0.995 0.98 0.95 0.90 Trichoderma harzianum was the most effective in inhibiting 25 ºC T. harzianum 444 X 12 the mycelial growth of Fusarium oxysporum f. sp. cubense N. oryzae 000 X 0 in vitro. Growth of F. oxysporum f. sp. ciceris was also sup- pressed by T. harzianum when these species were co-cul- 15 ºC T. harzianum 443 X 11 tured on PDA (Hervás et al., 1998, Dubey et al., 2007). N. oyzae 003 X 3 Biotic and abiotic parameters determine the extent of fungal colonisation and, among them, water activity, tem- I refers to sum of scores at 25 and 15 ºC for Trichoderma perature and fungal interactions are the most important harzianum competing with Nigrospora oryzae based on the (Torres et al., 2003). In this study, significant differences interaction scores for each species. Dominance on contact (4 for in water activity and temperature effects on growth rates the dominant species T. harzianum, 0 for the inhibited species were registered for T. harzianum and N. oryzae (P < 0.001) N. oryzae). Mutual antagonism at a distance (3 for both species). (X) Analysis of interaction was discarded. (Table 1). -1 -1 Growth rate (mm day ) Growth rate (mm day ) 10 F. Sempere and M.P. Santamarina 0.95 a 0.98 a 0.995 a w w w 0.95 a 0.98 a 0.995 a w w w FIG. 2 - Petri plates showing the interaction between Trichoderma harzianum (left) and Nigrospora oryzae (right) after 8 weeks at dif- ferent water activities and temperatures. Row A: 25 ºC. Culture of N. oryzae was inhibited by T. harzianum. Both species were growing until both colonies got in contact. Later T. harzianum grew trough N. oryzae. Row B: 15 ºC. The same type of interaction of Row A was registered (0.98 and 0.995 a ), while T. harzianum inhibited N. oryzae growth at a distance (0.95 a ), colony of T. harzianum. growth of T. harzianum was very similar in both situations Microscopic observation of the inoculation of N. oryzae (data not shown). three days later inoculation of T. harzianum revealed the Finally, the numerical values used in the Index of mechanism of mycoparasitism at all conditions tested Dominance revealed that T. harzianum was the dominant except at 0.95 a and 15 ºC. One of the earliest events of species over N. oryzae at 25 and 15 ºC (Table 2). the antagonistic process was the apparent affinity of T. harzianum for hypha and conidia of the N. oryzae (Fig. 3C Mycelial interactions between Trichoderma harzia- and 3D). However, how Trichoderma species “recognise” num and Nigrospora oryzae their host and attack host cells is yet unknown. This When T. harzianum and N. oryzae were inoculated at the process may involve hydrophobic interactions or interac- same time, conidia of N. oryzae were not observed (data tions between complementary molecules present on the not shown). Macroscopic examination of the Petri plates surface of both the host and the mycoparasite such as showed discoloured aerial mycelium of N. oryzae (Fig. 2). between lectins and carbohydrates (Whipps, 2001). These observations suggested that T. harzianum sup- Different researchers worked in this mycoparasite and oth- pressed sporulation of its host. ers species for elucidated this process and signalling path- Previously, this capacity was proposed as a potential ways that occur latter recognition of the host (Elad et al., strategy of biological control (Sutton and Peng, 1993; Köhl 1983; Inbar and Chet, 1994; Neethling and Nevalainen, and Fokkema, 1998). In different studies, the antagonist 1996; Omero et al., 1999). Clonostachys rosea reduced and suppressed conidia of Occasionally T. harzianum hyphae coiled around the Botrytis cinerea (Sutton et al., 1997; Morandi et al., 2003). hyphae and conidia of the pathogen from where penetra- This behaviour was also observed when mycoparasitic tion took place (Fig. 3E and 4D). Also, directly penetration Pythium oligandrum was confronted against Fusarium cul- of T. harzianum into hyphae and conidia of N. oryzae was morum in dual culture (Davanlou et al., 1999). Studies of observed (Fig. 3F, 4A, 4C, and 4D). Microscopic examina- dual cultures of T. harzianum and other species revealed tions showed loss of turgor and cell collapse of N. oryzae inhibition of germination but no suppression of conidia conidium (Fig. 3B) and disintegrated hyphal walls. Finally, (Lorito et al., 1993a, 1993b; Zimand et al., 1996; Kapat et T. harzianum utilised both their cell walls and cellular con- al., 1998; Golam Mortuza and Ilag, 1999). tents for nutrition and come out producing sexual struc- Individual culture of N. oryzae sporulated at all condi- tures (Fig. 3F, 4A, 4C, and 4D). tions tested (0.995, 0.98 and 0.95 a at 15 and 25 ºC). For The enzymes chitinases and glucanases from other strain belonging to the same species isolated from Trichoderma harzianum have been demonstrated to play the rice grains of Valencia, only formed conidia at 0.995 an important role in mycoparasitic action against different and 0.98 a at 25 ºC (Sempere and Santamarina, 2006b). fungi and others organisms (Viterbo et al., 2002; Ezziyyani This filamentous fungus formed unicellular solitary conidia, et al., 2004; Limón et al., 2004; Binod et al., 2007). globose or subglobose shaped with a smooth surface tex- However, fungal proteases may be significantly involved in ture, irregularly grown from short or ill-defined conidio- antagonistic activity, not only in the breakdown of the host phores (Fig. 3A and 3B). cell walls but also by inactivation of enzymes of pathogens Ann. Microbiol., 58 (1), 7-14 (2008) 11 A B 0.995 a , 15 ºC, X2000 0.995 a , 25 ºC, X3500 C D 0.98 a , 15 ºC, X2000 0.95 a , 25 ºC, X5000 w w D E 0.98 a , 25 ºC, X2000 0.995 a , 25 ºC ,X3500 w w FIG. 3 - Mycoparasitic interactions between Trichoderma harzianum and Nigrospora oryzae. A and B: conidia and conidiophores of N. oryzae, it can be seen their morphology still undamaged; C and D: conidia and hypha of N. oryzae in the beginning of being attacked by T. harzianum; E: hypha of T. harzianum coiling around hypha of N. oryzae (arrows); F: direct penetration of T. harzianum into partially disintegrated conidia of N. oryzae. A B 0.995 a , 15 ºC, X1000 0.98 a , 25 ºC, X7500 w w C D 0.95 a , 25 ºC, X2000 0.995 a , 25 ºC, X2000 w w FIG. 4 - Mycoparasitic interactions between Trichoderma harzianum and Nigrospora oryzae. A: directly penetration of T. harzianum into partially disintegrated conidia of N. oryzae (arrows); B: detail of the figure A, loss of turgor and cell collapse of N. oryzae conidium caused by T. harzianum; C: conidia of T. harzianum coming out of a degraded hypha of N. oryzae (arrow); D: hypha of T. harzianum coiling around, penetrating and coming out a conidium of N. oryzae. 12 F. Sempere and M.P. Santamarina nd (Rodriguez-Kabana et al., 1978, Suarez et al., 2004). Diseases of Cereals and Pulses, 2 edn., Prentice-Hall Inc., USA, pp. 116-129. Proteolytic enzymes of T. harzianum reduced Botrytis cinerea germination and deactivated partially hydrolytic Cook R.J. (1993). Making greater use of introduced microorgan- isms for biological control of plant pathogens. Annu. Rev. enzymes endo-PG and exo-PG produced by this strain (Elad Phytopathol., 31: 53-80. and Kapat, 1999). Cryo-SEM study in the interaction zone, Davanlou M., Madsen A.M., Madsen C.H., Hockenhull J. 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Evaluation of Trichoderma species against Fusarium oxysporum f. sp. Trichoderma pseudokoningii against Botryodiplodia theo- ciceris for integrated management of chickpea wilt. Biol. bromae. Antagonism at a distance has been usually attrib- Control, 40: 118-127. uted to diffusible or volatile compounds (Sonnenbichler et Elad Y., Barak R., Chet I. (1983). Possible role of lectins in myco- al., 1994). These morphological alterations of the pathogen parasitism. J. Bacteriol., 154 (3): 1431-1435. and its inhibition they could be due to the secretion of these Elad Y., Kapat A. (1999). Role of Trichoderma harzianum pro- substances by T. harzianum. In this way, species of tease in the biocontrol of Botrytis cinerea. Eur. J. Plant Trichoderma, which are currently all registered biological Pathol., 105: 177-189. control products, are known to produce several secondary Ezziyyani M., Pérez C., Ahmed A.S., Requena M.E., Candela M.E. metabolites inhibitory to the growth of the host with broad- (2004). Trichoderma harzianum como biofungicida para el biocontrol de Phytophthora capsici en plantas de pimiento spectrum antimicrobial activity (Dennis and Wester, 1971a, (Capsicum annuum L.). Anales de Biología, 26: 35-45. 1971b; Punja and Utkhede, 2003). Cryo-SEM has been proved a useful technique to Fresco L. (2005). ‘‘Rice is life’’. J. Food Comp. Anal., 18: 249- observe and describe different interactions between Gokulapalan C., Nair M.C. (1986). Mycoparasites of Rhizoctonia microorganisms. Previously the authors investigated other solani and control of sheath blight of rice, (abstract) interactions and all studies confirmed this technique. There Fourteenth Annual Meeting of the Mycological Society of are few researches and the usual techniques to study fun- India and Seminar on Applied Micology, Thanjavur, India, p. gal relations are tedious SEM preparations or light micro- scopical observations (Madi et al., 1997; Davanlou et al., Golam Mortuza M., Ilag L.L. (1999). Potential for biocontrol of 1999; Moussa, 2002; Zadworny et al., 2004). Lasiodiplodia theobromae (Pat.) Griff. & Maubl. in banana fruits by Trichoderma species. Biol. Control, 15: 235-240. Knowledge of the mechanism of action involved in the biocontrol process can permit establishment of optimum Gupta V.P., Tewari S.K., Govindaiah, Bajpai A.K. (1999). Ultrastructure of mycoparasitism of Trichoderma, conditions for the interaction between the pathogen and Gliocladium and Laetisaria species on Botryodiplodia theo- the biological control agent and is important for imple- bromae. J. 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