Identification of Dominant Trichoderma Species in Pistachio Orchards of Kerman Province

پژوهش های حفاظت گیاهان ایران

Article 10, Volume 30, Issue 1 - Serial Number 31, March 2016, Pages 82-92   

Document Type: Research Article

DOI: 10.22067/jpp.v30i1.31803

Authors

F. Mirkhani^* ; H. Alaei; Amir hossein mohammadi; M. Haghdel

Pistachio Research Center, Agricultural Research, Education and Extension Organization

Abstract

Introduction: Trichoderma species are significant biocontrol agents against fungal plant pathogens. Some species produce a wide diversity of metabolites, antibiotics, as well as the toxins. Because of the intimate relationship between species of Trichoderma and human activity, there is a great need for the accurate identification of Trichoderma species. Accurate species identification is possible only through a combination of morphological and molecular methods. For identification of new Trichoderma species, most authors have used the combination of ITS and tef1. Many studies have been done on the taxonomy of this genus in the world. Nevertheless, the information about the diversity of Trichoderma/Hypocrea in Iran is scarce. Due to this, in discussing the use of antagonistic agents in biological control, the establishment of biocontrol agents seems to be the first and most important step. Thus, identifying effective native biocontrol agents to be used against plant pathogen agents in an area is of considerable importance. There are no reports for the biodiversity of Trichoderma species on saline and alkaline soils of pistachio orchards in Iran and the world. Because of the alkaline and saline nature of Iranian pistachio soils and the importance and applications of Trichoderma species in biological control of plant pathogens, the objective of the present study was to evaluate the occurrence and species diversity of Trichoderma isolates recovered from alkaline soils of pistachio orchards based on morphological and molecular analysis.
Materials and Methods: Soil samples were collected from a depth of 0 – 60 cm the rhizosphere of pistachio trees in Kerman province during 2010 – 2012. The samples were then transferred to the laboratory. Trichoderma species were isolated from soil samples using a selective Trichoderma medium (TSMC). The pH and electrical conductivity EC of each soil sample were measured. The morphological identification of Trichoderma isolates was performed based on macro- micromorphological observation, including rate growth and colony formation, presence or absence of pustules, pigmentation and sporulation, as well as conidiophores shape and branching, size and shape of phialid, conidia and chlamydospores on PDA and CMD media using valid Trichoderma keys. Then, the Trichoderma isolates were grouped in the preliminary experiment based on morphological characteristics and the resulted data from RAPD molecular marker using the primer A-5. The molecular identification of selected Trichoderma isolates was performed based on determination of the complete sequences of ribosomal DNA internal transcribed spacer regions using the primer set ITS1F-ITS4 and identified by submission to GenBank and the BLAST interface in TrichoKEY 2.0 to BLAST homology search.
Results and Discussion: In our present study, 194 soil samples were collected from pistachio orchards. The soils were near neutral to alkaline with an optimal pH range of 7.0 to 8.3 and the EC ranged from 1.5 to12.3 dSm–1. A total of 117 isolates of Trichoderma were obtained and purified. Isolates were identified to species level by a combination of morphological and genotypic characters. Before molecular identification, the Trichoderma isolates were grouped according to an examination of their morphology on PDA and CMD, using the following macroscopic and microscopic characteristics. Also, all isolates of each group were analyzed by RAPD molecular marker using the primer A-5. The RAPD-PCR profile analysis showed 16 different band patterns. The representative isolates of each profile was selected for DNA sequencing. As a result the isolates were identified as four species including T. harzianum, T. virens, T. brevicompactum and T. longibrachiatum. The results showed that more than 50% of the isolates belonged to T. harzianum and introduced as the dominant species in soil of pistachio orchards in Kerman province. Based on logistic regression analysis, although there was no relationship between the soil properties (pH, EC) and the presence of Trichoderma (R2=0.25, Pr=0.74, 0.26>0.05) but decreasing in numbers and diversity of Thrichoderma species in testing soils could be a result of alkaline conditions. To our knowledge, this study was the first report of identification and diversity of Trichoderma species in alkaline soils and pistachio trees.
Conclusion: A low degree of biodiversity of Trichoderma isolates was found in soils of pistachio orchards. In fact, acidity and alkaline conditions as well as environmental parameters such as soil temperature, moisture, pH, organic matter (OM) and nutrient content are factors that could affect on Trichoderma population, such as its presence, density, longevity as well as production of enzymes.

Keywords

Logistic Regression; morphology; Ribosomal DNA; Trichoderma

References

1- Akrami M., Golzary H., and Ahmadzadeh M. 2011. Evaluation of different combinations of Trichoderma species for controlling Fusarium rot of lentil. African Journal of Biotechnology, 10: 2653-2658.

2- Alaei H., Baeyen S., Maes M., Hofte M., and Heungens K. 2009. Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR. Journal of Microbiological Methods, 76:136-145.

3- Anees M., Tronsmo A., Edel-Hermann V., Hjeljord L.G., Heraud C., and Steinberg C. 2010. Characterization of field isolates of Trichoderma antagonistic against Rhizoctonia solani. Fungal Biology, 114:691-701.

4- Bazgir E., Rouhani H., and Okhovat M. 1991. Evaluation of antagonistic effect of Gliocladium sp. on Rhizoctonia solani the causal agent of bean damping-off and seed rot. p. 108. Proceeding of the 10th plant protection congress of Iran, 1-5 sep. 1991. Kerman, Iran.

5- Bissett J. 1984. A revision of the genus Trichoderma. I. Section Longibrachiatum sect. Nov. Canadian Journal of Botany, 62:924-931.

6- Bissett J. 1991a. A revision of the genus Trichoderma. II. Infrageneric classification. Canadian Journal of Botany, 69:2357-2372.

7- Bissett J. 1991b. A revision of the genus Trichoderma. III. Section Pachybasium. Canadian Journal of Botany, 69:2373- 2417.

8- Bissett J. 1991c. A revision of the genus Trichoderma. IV. Additional notes on section Longibrachiatum. Canadian Journal of Botany, 69:2418-2420.

9- Bissett J., Szakacs G., Nolan C.A., Druzhinina I., Gradinger C., and Kubicek C.P. 2003. New species of Trichoderma from Asia. Canadian Journal of Botany, 81:570-586.

10- Blaszczyk L., Popiel D., Chelkowski J., Koczyk G., Samuels G.J., Sobieralski K., and Siwulski M. 2011. Species diversity of Trichoderma in Poland. Journal of Applied Genetics, 52:233-243.

11- Bourguignon, E. 2008. Ecology and diversity of indigenous Trichoderma species in vegetable cropping systems. Ph.D dissertation, National Centre for Advanced Bio-Protection Technologies, Lincoln University.

12- Campos, A.R.M. 2012. Isolation native strain of Trichoderma spp from soil horticulture tiluca valley with potentailof biocontrol on Sclerotinia. Tropical and Subtropical Agroecosystems, 15:357-365.

13- Chacraborty B.N., Chacraborty U., Saha A., Dey P.L., and Sunar K. 2010. Molecular characterization of Trichoderma viride and Trichoderma harzianum isolated from soils of North Bengal based on rDNA markers and analysis of their PCR-RAPD profiles. Global Journal of Biotechnology and Biochemistry, 5:55-61.

14- Druzhinina I.S., Kopchinskiy A.G., Komon M., Bissett J., Szakacs G., and Kubicek C.P. 2005. An oligonucleotide barcode for species identification in Trichoderma and Hypocrea. Fungal Genetics and Biology, 42:813-828.

15- Druzhinina I.S., and Kubicek C.P. 2005. Species concepts and biodiversity in Trichoderma and Hypocrea: from aggregate species to species clusters? Journal Zhejiang University Science, 6(2):100-112.

16- Ehyaie A., and Behbahanizade A.A. 1372. The methods of soil chemical analysis. Soil and Water Research Institute, Technical Bulletin.

17- Elad Y., and Chet I. 1983. Improved selective media for isolation of Trichoderma spp. or Fusarium spp. Phytoparasitica, 11:55-58.

18- Ershad D. 2009. Fungi of Iran. Iranian Ministry of Agriculture, Tehran, Iran.

19- Gams W., and Bissett J. 1998. Morphology and identification of Trichoderma. p. 3-34. In Trichoderma and Gliocladium. Vol. 1. Basic Biology, Taxonomy and Genetics. Taylor and Francis Ltd. London.

20- Garbeva P., Van Veen J.A., and Van Elsas, J.D. 2004. Microbial diversity in soil: selection of microbial populations by plant and soil type and implications for disease suppressiveness. Annual Review Phytopathology, 42:243-270.

21- Gardes M., and Bruns T.D. 1993. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Molecular Ecology, 2:113-118.

22- Gherbawy Y., Druzhinina I.S., Shaban G.M., Wuczkowsky M., Yaser M., El-Naghy M.A., Prillinger H.J., and Kubicek C.P. 2004. Trichoderma populations from alkaline agricultural soil in the Nile valley, Egypt, consist of only two species. Mycological Progress, 3:211-218.

23- Hoyos-Carvajal L., Orduz S., and Bissett J. 2009. Genetic and metabolic biodiversity of Trichoderma from Colombia and adjacent neotropic regions. Fungal Genetics and Biology, 46:615-631.

24- Isnaini M., Burgess D.R., and Keane P.J. 1998. The use of cultures of Sclerotinia minor for selective isolation and enumeration of mycoparasitic isolates of Trichoderma from soil and roots. Australasian Plant Pathology, 27:244-250.

25- Johnson L.F. 1959. Methods for Studying Soil Microflora Plant Disease Relationships. Minneapolis, Burgess Publishing Company.

26- Kopchinskiy A., Komon M., Kubicek C.P., and Druzhinina I.S. 2005. Tricho blast: a multilocus database for Trichoderma and Hypocrea identifications. Mycological Research, 109:658-660.

27- Kraus G.F., Druzhinina I.S., Gams W., Bissett J., Zafari D., Szakacs G., Kopchinskiy A., Prillinger H., Zare R., and Kubicek C.P. 2004. Trichoderma brevicompactum sp. nov. Mycologia, 96:1059-1073.

28- Kredics L., Antal Z., Manczinger L., Szekeres A., Kevei F., and Nagy E. 2003. Influence of environmental parameters on Trichoderma strains with biocontrol potential. Food Technology and Biotechnology, 41:37-42.

29- Kubicek C.P., Bissett J., Druzhinina I.S., Kullnig-Gradinger C., and Szakacs G. 2003. Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates. Fungal Genetics and Biology, 38:310-319.

30- Kubicek C.P., and Penttila M.E. 1998. Regulation of production of plant polysaccharide degrading enzymes by Trichoderma. p. 49-71. In G.E. Harman and C.P. Kubicek (eds.) Trichoderma and Gliocladium: Enzymes, Biological Control and Commercial Applications. Taylor and Francis Ltd. London, UK.

31- Kubicek C.P., Komon-Zelazowska M., and Druzhinina I.S. 2008. Fungal genus Hypocrea/Trichoderma: from barcodes to biodiversity. Journal of Zhejiang University-Science B, 9:753-763.

32- Kullnig C., Szakacs G., and Kubicek C.P. 2000. Molecular identification of Trichoderma species from Russia, Siberia and the Himalaya. Mycological Research, 104:1117-1125.

33- Leandro L.F.S., Guzman T., Ferguson L.M., Fernandez G.E., and Louws F.J. 2007. Population dynamics of Trichoderma in fumigated and compost-amended soil and on strawberry roots. Applied Soil Ecology, 35:237-246.

34- Lopez-Mondejar R., Ros M., and Pascual J.A. 2011. Mycoparasitism-related genes expression of Trichoderma harzianum isolates to evaluate their efficacy as biological control agent. Biological Control, 56:59-66.

35- Michel-Aceves A.C., Rebolledo-Dominguez O., Lezama-Gutierrez R., Ochoa-Moreno M.E., and Samuels G.J. 2001. Especies de Trichoderma en suelos cultivados con mangoafectador por Escoba de bruja y su potencial inhibitorio sobre Fusarium oxysporum y F. subglutinans. Revista Mexicana de Fitopatologia, 19:154-160.

36- Miller S.L. 1957. The mechanism of synthesis of amino acids by electric discharges. Biochimica et Biophysica Acta, 23:480-489.

37- Migheli Q., Balmas V., Komon-Zelazowska M., Scherm B., Fiori S., Kopchinskiy A.G., Kubicek C.P., and Druzhinina I.S. 2009. Soils of a mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/Trichoderma. Environmental Microbiology, 11:35-46.

38- Naeimi S., Okhovat M., Javannikkhah M., Kredics L., and Khosravi V. 2010. Frequency and distribution of Trichoderma spp. in the rice (paddy) fields of Mazandaran province, Iran. Iranian Journal of Plant Protection Science, 40: 79-91. (in Persian with English abstract).

39- Nazmi Roudsari F., Zafari D., Khodaparast S.A., and Rouhani H. 2007. Introducing some new species of Trichoderma for Iran. Rostaniha, 8: 67-83. (in Persian with English abstract).

40- Okoth S.A., Roimen H., Mutsotso B., Muya E., Kahindi J., Owino J.O., and Okoth P. 2007b. Land use systems and distribution of Trichoderma species in Embu region, Kenya tropical and subtropical agroecosystems. Tropical and Subtropical Agroecosystems, 7:105-122.

41- RezaeeDanesh U.R., Mohammadi Goltapeh A., and Rouhani H. 2000. Identification of Trichoderma species causing green mold in button mushroom farms, and their loss assessment. p. 104. Proceedings of the 14th Iranian Plant Protection Congress, vol. II, 5-8 Sep. 2000. Isfahan, Iran.

42- Rifai M.A. 1969. A revision of the genus Trichoderma. Mycological Papers, 116:1-116.

43- Rouhani H., and Safari M. 1998. Introducing species of Trichoderma Pers. from Iran. p. 301. Proceedings of the 13th Iranian Plant Protection Congress, vol. II, 23-27 Aug. 1998. Karaj, Iran.

44- White T.J., Bruns T., Lee S., and Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: a guide to methods and applications, 18:315-322.

45- Wuczkowski M., Druzhinina I.S., Gherbawy Y., Klug B., Prillinger H., and Kubicek C.P. 2003. Species pattern and genetic diversity of Trichoderma in a mid-European, primeval floodplain-forest. Microbiological Research, 158:125-133.

46- Zafari D., Ershad D., Zare R., and Alizadeh A. 2002. A contribution to the identification of Trichoderma species in Iran. Iranian Journal of Plant Pathology, 38: 21-45. (in Persian with English abstract).

47- Zafari D., Ershad D., Zare R., and Alizadeh A. 2004. Three new species of Trichoderma for the mycoflora of Iran. Rostaniha, 5: 159-170. (in Persian with English abstract).

48- Zhang C., Druzhinina I.S., Kubicek C.P., and Xu T. 2005. Trichoderma biodiversity in China: evidence for a North to South distribution of species in East Asia. FEMS Microbiology Letters, 251:251-257.