Clonostachys rosea, a new and promising entomopathogenic fungus infecting pupa of jujube fruit fly, Carpomya vesuviana

Document Type: Original Article


1 Plant Protection Research Department, South Khorasan Agricultural and Natural Resources Research and Education Center, AREEO, Birjand, Iran

2 Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran


Jujube fruit fly, Carpomyia vesuviana, is the most important pest of jujube in Iran. During February and March 2013, a survey on fungi associated with pupae of C. vesuviana was conducted in Birjand, South Khorasan province of Iran. To isolate fungi, the samples were washed under running tap water, surface-sterilized in 0.5% sodium hypochlorite, then rinsed and placed onto potato dextrose agar. 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 the morphological and molecular studies, based on ITS-rDNA. Both morphological and molecular data confirmed the identity of this fungus as Clonostachys rosea. Based on pathogenicity assays, significant mortality of inoculated pupa (46%) was recorded with a conidial suspension of 1010 (spore/ ml). Median Lethal Time (LT50) and LC50 values of this isolate against pupa of C. vesuviana were calculated 4.6 days and 5.1×104 spore/ml, respectively. A significant positive correlation between different 3 C. rosea concentrations and mortality of C. vesuviana was observed (r2= 0.9986, p= 0.003). Clonostachys rosea is here reported as a newand promising entomopathogenic fungus on C. vesuviana, suggesting that it is likely to play roles in protecting jujube trees against this pest.


Main Subjects


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.



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:// 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).



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). 

Table.1. The taxa were used in the phylogenetic analysis.





GenBank accession no. (ITS)


Clonostachys rosea

IRAN 3253C

Carpomyia vesuviana



This study

Bionectria ochroleuca

BBA 68698




Hagedorn and Scholler 1999

Clonostachys sporodochialis



French Guiana


Lechat et al. 2014

Bionectria ochroleuca

CBS 710.86




Schroers 2001

Clonostachys rosea


Bemisia tabaci



Anwar 2016

Bionectria levigata

CBS 948.97

Buxus sempervirens



Schroers 2001

The sequence written in bold is generated in this study. CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; IRAN: Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Tehran, Iran; LPSC: La Plata Spegazzini Collection, Argentina.


Conidia from both types of conidiophores were hyaline, smooth-walled, slightly curved, 4–7 × 2–3.1 µm (fig. 1-e). Based on the above-mentioned characteristics, the fungus was identified as Clonostachys rosea. The phylogenetic analyses performed using ITS region sequences including our isolate and the others obtained from GenBank. The phylogenetic tree showed that the examined isolate belonging to a moderately supported clade in NJ analysis (support value= 72), is nested with some strains of C. rosea (Fig. 2). 

Pupae inoculated with C. rosea were infected (Fig 1-a). The fungus re-isolated from inoculated pupa was identical to the original isolates, confirming Koch's postulates. The LC50 value was calculated by 5.1×104 spore/ml. Statistical analysis showed a significant difference between the different spore concentrations of the C. rosea and mortality of C. vesuviana (p<0.001). Mortality of inoculated pupa with 1010 (spore/ml) was recorded 46% compared with control (Table 2). A significant positive correlation between concentrations and mortality was observed (r2 = 0.9986, p = 0.003) (Fig. 3). Median lethal time (LT50) values of this isolate against pupa of C. vesuviana was calculated 4.6 days and ranged from 3.1 to 6.1 days (Table 3).

Fig. 1. a. Infected pupae of Carpomya vesuviana by Clonostachys rosea in pathogenicity tests, b. naturally infected pupae of C. vesuviana showing fungal structure of C. roseac. secondary penicillate conidiophores, d. primaryVerticillium-like conidiophores of C. roseae. conidia.

Fig. 2. Neighbor-joining tree based on ITS-rDNA sequences of Clonostachys rosea. Bootstrap values >50% (1000 replicates) shown at the nodes. Bionectria levigata serves as outgroup.

Fig. 3. Effect of different conidial concentrations of Clonostachys rosea on the mortality of pupa Carpomya vesuviana after 7 days.



The taxonomic characteristics of the fungus described in this study were consistent with those reported by Schroers (2001) and Toledo et al. (2006). The destructive, necrotrophic, and unspecific mycoparasitism of C. rosea was first described by Barnett and Lilly (1962). Clonostachys rosea has been isolated from various plant substrates by Schroers (2001). This research proposed that species of Bionectria/Clonostachys do not have serious plant-pathogenic abilities. As a mycoparasite, C. rosea was successfully tested as a biological control agent of gray mold, caused by Botrytis cinerea Pers. (Cota et al. 2008). A basic formulation of C. rosea strain ACM941 has been applied against Fusarium head blight of wheat and root rot of soybeans in the field but there are few reports about entomopathogenic efficacy of this fungus. Toledo et al. (2006) reported C. rosea as an entomopathogenic fungus of Oncometopia tucumana (Schröder) and Sonesimia grossa (Signoret) in Argentina. Pathogenicity of C. rosea against coffee berry borer, Hypothenemus hampei (Ferrari), has been reported by Vega et al. (2008). Successful control of Delia radicum (Linnaeus), an important pest of Brassicaceous crops, using Clonostachys solani f. nigrovirens (J.F.H. Beyma) Schroers has been reported by Razinger et al. (2014).

The results obtained by this study showed that there is a high parasitic rate with increasing conidia concentration of C. rosea on pupa of C. vesuviana. Anwar et al. (2018) also showed a reduction in mortality B. tabaci nymphs by C. rosea at the lower spore concentrations. Our results showed that the examined C. rosea isolate leading to 46% pupae jujube fruit fly mortality 7 days after inoculation. Toledo et al. (2006) also observed 82.5% mortality of Oncometopia tucumana after 14 days of inoculation using C. rosea isolates. In similar research, Mustafa et al (2014) reported 55.1% mortality of Alfalfa weevil 6 days post-inoculation by C. rosea.


Table 2. Effect of conidial concentrations of Clonostachys rosea (IRAN 3253C) against Carpomya vesuviana.


(per ml)

No. of pupa


Number of infected pupae after spore spraying (day)

Mortality (%) *



























































* Mean followed by the same letter not significantly different

Table 3. LT50 values of conidial suspension of Clonostachys rosea (1 × 1010 conidia/ml) on pupa of Carpomya vesuviana*

Fungal Isolate

LT50 (days)

95% confidence limits

Lower limit

Upper limit

IRAN 3253C





Several biocontrol mechanisms have been reported for C. rosea as a mycoparasite or Entomopathogenic agent. Zhang et al. (2008) reported that the pathogenesis started from the adherence of conidia to nematode cuticle for germination, followed by the penetration of germ tubes into the nematode body and subsequent death and degradation of the nematodes. Direct parasitism, production of fungal cell wall degrading enzymes, antibiosis, induction of plant defense reactions and toxic metabolites production by this fungus have been reported as biocontrol mechanisms of C. rosea (Nygren et al. 2018). This fungus been considered as an entomopathogenic fungus and therefore may contribute to biological control of insects (Keyser et al. 2016). To our knowledge, this is the first record of C. rosea on jujube fly throughout the world. As the pest overwinters as a pupa in the soil (Shan Yong et al. 2009), it has the potential to be considered as a biocontrol agent of the ber fruit fly pupae in the soil. Therefore, it seems that this fungus is likely to play roles in protecting jujube trees against pests and also promoting plant growth simultaneously. However, further research on its efficiency as a biocontrol agent or as an endophyte is required.



With an emphasis on organic production of the jujube, biological control of jujube fruit fly (the most important pest of jujube in Iran) provides opportunities for reducing negative impacts of chemical pesticides on the environment, biodiversity and human health. In this study, C. rosea is reported as a potential biological control agent of jujube fruit fly, C. vesuviana. However, further studies are recommended to evaluate its potential in field conditions, and determining its presumably endophytic role.



This study is a part of the project "Isolation of bacterial and fungal antagonist of jujube flies” that is implemented at South Khorasan Agricultural and Natural Resources Research and Education Center, Iran.

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