Identification of Dominant Trichoderma Species in Pistachio Orchards of Kerman Province

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.
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.
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.
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.