INTRODUCTION

Jujube (Zizyphus jujuba Mill.), belonging to the family Rhamnaceae, is distributed in the warm temperate and sub-tropical regions of the world (Mirzaee et al. 2013). The jujube fruit fly, Carpomyia vesuvianaCosta (Diptera: Tephritidae) is the most important pest of jujube in Iran (Moodi 2002), causing considerable damage to this crop at the fruiting stage. A variety of control methods including chemical pesticides, cultural practices, resistant cultivars, and attractant agents have been used for its control in Iran (Tavakoli Korghond et al. 2012). However, an attractive alternative method to control pests of agricultural crops is biological control. Microbial biocontrol agents (MBCAs) are as the natural enemies of the pest population that have no hazard effects on human health and the environment. Application of non-chemical entomopathogenic agents for control of some pests have been conducted and recommended. For example, Andreev et al. (2008) suggested Beauveria bassiana (Bals.-Criv.) Vuill. as the best agent for control of black cherry aphid, Myzus cerasi (Fabricius). Besides, Sabbour et al. (2012) have evaluated the efficiency of Beauveria bassiana, Metarhizium anisopliae (Metschn.) Sorokin and Isaria fumosorosea Wize against three important stored product pests, Plodia interpunctella (Hubner), Ephestia cautella (Walker) and E. kuehniella Zeller. Due to their environmental safety and specificity, bio-pesticides are gaining popularity in the management of pests of horticultural crops. Clonostachys rosea (Link) Schroers, Samuels, Seifert & W. Gams (teleomorph: Bionectria ochroleuca (Schwein.) Schroers & Samuels) has been reported as a potential parasite of insects (Toledo et al. 2006) and nematodes such as Heterodera spp. and Globodera spp. (Silva et al. 2015). This fungus also has been reported as pathogenic on sclerotia of Sclerotinia sclerotiorum (Lib.) de Bary, Phymatotrichum omnivorum Duggar, Rhizoctonia solani J.G. Kühn, Botrytis spp. and Verticillium spp. (Sutton et al. 1997). This widely distributed fungal species, often naturally found in agricultural soils, is a promising effective biological control agent (Keyser et al. 2016; Sun et al. 2017). This study describes a fungal antagonistic agent of the jujube fruit fly as the first step of the biological control program of this pest.

MATERIALS AND METHODS

During surveys on jujube orchards in Birjand, South Khorasan province of Iran in February and March 2013, the pupae of jujube fruit fly showing symptoms of infection and emerging fungal structures were observed in the soil under trees. These pupae were collected in sterile Petri dishes and transferred to the laboratory. To isolate fungi, the infected pupae (fig1-b) were washed under running tap water, surface-sterilized in 0.5% (v/v) sodium hypochlorite, rinsed several times in sterile distilled water, placed onto potato dextrose agar (PDA, Merck), and incubated at 25°C in the dark. Out of eight isolates of Clonostachys rosea obtained in this study, only one showed considerable pathogenicity against the pupa of C. vesuviana. This isolate was additionally examined using morphological and molecular studies. Measurement of fungal structures (conidiophores, phialides and conidia) was based on single spore cultures on both PDA and Oat Meal Agar media (OMA, East Molesey). The pathogenic strain of C. rosea on pupae of jujube fruit fly is preserved at the Iranian Fungal Culture Collection (IRAN 3253C), Iranian Research Institute of Plant Protection (Tehran, Iran).

For pathogenicity assays and LC50 and LT₅₀ calculation of the representative strain of C. rosea, thirty individuals of jujube fly pupa were exposed to different conidial suspension of 10³, 10⁵, 10⁷, 10¹⁰ spore/ml, prepared from 10-day-old cultures grown on PDA at 25°C, for 10 sec and placed on sterile Petri dishes containing filter paper dampened with sterile distilled water. Each of 10 pupae was used for each concentration at three replicates. The control was treated with sterile distilled water. Treated and control pupae were maintained at 25°C. Percentage of pupal colonization was recorded after 14 days. Probit program of SAS (version 9.2) software was used to determine the concentration of the suspension of the fungus that killed 50% (LC50) of the examined pupa.

Genomic DNA of the representative strain of C. rosea was extracted using a modified Chelex 100 chelating resin method with an initial step of grinding the mycelia in liquid nitrogen (Walsh et al. 1991). The very small mycelium was transferred to a sterile micro-tube and crushed with liquid nitrogen. The crushed mycelium was transferred into a tube containing 200 µl of 5% w/v Chelex 100 resin suspension. The tubes were vortexed and incubated at 56°C overnight, then at boiling water bath for 7 min. The extracts were thoroughly vortexed, then incubated again in a boiling water bath for 8 min and centrifuged at 12,000 rpm for 5 min. The supernatant was transferred to a new sterile tube and diluted with TAE buffer (1:10). PCR amplification of the internal transcribed spacer (ITS) of the causal fungus was carried out using the primers of ITS4 and ITS5 (White et al., 1990). PCR was performed in a 25 μl volume reaction mixture with 1 μl Mgcl2, 2.5 μl PCR buffer (10X), 0.5μl dNTP mix, 1 μl of each primer, 0.4 μl Enzyme taq polymerase and 13.6 ml sterile water and 5μl DNA template. The amplification was performed on Eppendorf Thermo-cycler according to the following profile: an initial denaturation step at 94°C for 3 min, followed by 30 amplification cycles at 94°C for 45 sec, 55°C for 45 sec, 72°C for 2 min and a final extension step at 72°C for 7 min. The PCR product was detected by agarose gel electrophoresis and was visualized by ultraviolet (UV) fluorescence after the novel juice (Gene DireX) staining. PCR products sequenced by Macrogen Company (South Korea) and deposited in GenBank (NCBI) database (www.ncbi.nlm.nih. gov/genbank/) with accession number MK005284. The sequences were assembled and edited by MEGA software version 6 (Schroers. 2001). BLAST searches were done using the NCBI server (http:// www.ncbi.nlm.nih.gov/blast). Sequence alignments were generated by CLUSTALW implemented in MEGA software version 6 (Tamura et al. 2013). The phylogenetic tree was constructed using the neighbor-joining (NJ) method using the Tamura–Nei model (Tamura and Nei 1993) in MEGA 6.0. The robustness of the resulting tree was tested by bootstrapping with 1000 re-samplings of the data (Saitou and Nei 1987). Bionectria levigata (AF210680) was used as outgroup (table 1).

RESULTS

Eight strains of Clonostachys rosea were isolated from which the only one presented considerable pathogenicity on pupa of Carpomya vesuviana. This isolate (IRAN 3253C) was additionally examined using morphological and molecular studies. The microscopic and macroscopic observations were made from potato-dextrose agar (PDA) and oatmeal agar (OA) cultures as described by Schroers et al. (2001). Colonies were white to light yellow, producing orange pigments on cultures. The colony diam. reached 38–40 mm after 7d at 28 °C. The fungal isolate was characterized by two kinds of conidiophores. Primary conidiophores were verticillium-like, bearing phialides measuring 8–12.5 × 1.2–1.6 µm (n=100) (Fig 1-d), and secondary conidiophores were penicillate (Fig. 1-c).