3 19 Hasan, et al Bull. Iraq nat. Hist. Mus. (2012) 12 (1): 19-27 OCCURRENCE OF ENTOMOPATHOGENIC AND OTHER OPPORTUNISTIC FUNGI IN SOIL COLLECTED FROM INSECT HIBERNATION SITES AND EVALUATION OF THEIR ENTOMOPATHOGENIC POTENTIAL Wazeer A. Hasan*, Lazgeen Haji Assaf* and Samir Khalaf Abdullah** *Plant Protection Department, Faculty of Agriculture, School of Plant Production, Department of Plant Protection, Duhok University, Iraq **Biology Department, Faculty of Science, Zakho University,Duhok, Iraq. ABSTRACT A survey of entomopathogenic and other opportunistic fungi isolated from soil samples collected from insect hibernation sites in different habitats in Kurdistan region of Iraq was carried out during October to December 2009. By using dilution plate method, two entomopathogenic species (Beauveria bassiana (Bals.) Vuill.and Isaria javanica (Friedrichs & Bally) Samson & Hywel-Jones) were detected with isolation percentage (38.46%) each. Other opportunistic fungi such as Alternaria alternata, Aspergillus flavus, A.niger, Penicillium glabrum, P. digitatum, Rhizopus stolonifer and Syncephalastratum racemosum were also isolated. B. bassiana was the most virulent fungus and showed complete mortality (100%) on two aphid species Hyalopterus pruni Geoff. and Aphis pomi De Greer after six days of inoculation, followed by I.javanica with 66.67% and 75.59% mortality respectively. I. javanica was isolated for the first time from Iraq. A brief description along with photographs is provided for the newly recorded species. Key words: Entomopathogenic fungi, Soil, Iraq. Correspondence; Samer_abdalh@yahoo.com INTRODUCTION Entomopathogenic fungi were occurred naturally as infections in insect or arachid hosts, and several of these fungi only occurred as infections in living hosts for a relatively short period of time during their life cycle. The remainder of the life cycle of these species presumably lurk as dormant propagules in the soil, in the vicinity of the dead host cadaver. Thus, the chances of finding good candidates to be used as biocontrol agents in these soils are very high (Olivares-Bernabeu and Lopez-Llorca, 2002). Most fungi from the order Hypocreals are only known in their anamorphic life cycle, thus only mitosporic conidia are formed. The dead host cadavers will mostly fall to the ground, and thus, a reservoir of fungal material is present in the soil environment. Further, dispersal from cadavers as focal points presumably occur due to weather (wind and rain), soil manipulation and also insect activity (Meyling et al., 2006). Soil factors (temperature, pH, or organic content, relative moisture or mineral, organic or biotic factors) can affect fungal persistence and activity (Charnley, 1997). In the laboratory, however, the conidia from hypocrelean entomopathogenic fungi can also germinate and grow on artificial media, and need to come in contact with susceptible host in mailto:Samer_abdalh@yahoo.com 20 Occurrence of Entomopathogenic order to grow and proliferate successfully. These two methods of germination are manipulated for isolation of entomopathogenic fungi from the soil ( Goettel and Inglis,1997). In order to monitor the fate of applied fungal material in the soil, a selective media originally described by Strasser et al. (1996) were used for detection of survival Beauveria spp. Metarhizium spp., and Paecilomyces spp., Bacteria can be inhibited by the application of broad-spectrum antibiotics such as chloramphenicol, tetracycline, or streptomycin (Goettel and Inglis, 1997). However, to exploit the ability of the fungi to infect host, the insect bait method can be used (Zimmermann, 1986). In the present work we have analyzed the presence of entomopathogens, mainly fungi, in soil samples collected from insect hibernation sites in different ecosystems of Duhok province such as natural forests of Quercus rotundifolia, agricultural soils and grape orchards and to evaluate their entomopathogenic potential on two aphid species. MATERIALS AND METHODS Sampling sites and Colleton of soil samples Thirteen soil samples were collected from three insect hibernation sites in Duhok province,North Iraq during October to December 2009. The sites included fields with agricultural soil at Barway Bala (4 sampls), natural forests mainly Qurcus rotundifolia at Gara mountain (6 samples) and from grapevine yard at Siara Tooka (3 samples). Soil samples about (500 g each) were taken from the depth of 0-10 cm with a trowel after removing litter or weed plants that insects hidden beneath then, placed in plastic bags and brought to the laboratory. Samples were subjected for fungal isolation within 2 days of collection. Fungal isolation and identification: Initial dilution was made by mixing 10g of soil with 90 ml of sterilized distilled water in 250 ml conical flask. Flasks were shaken for 3 minutes on an electrical shaker. Serial dilutions up to 104 were made in the same method. One ml. of 104 dilution was poured in each plate and mixed with 20 ml. of Potato dextrose agar medium (Himedia laboratories,Ltd. India) supplemented with 0.28 mg/l chloramphenicol to avoid bacterial growth. Six plates per replication were used. The plates were incubated at 25 C for 7 days. The isolates were purified and growing colonies were identified depending on their morphological characteristics of their reproductive structures with the aid of several taxonomic references (Samson 1974; Domsch et al.1980; Goettel and Inglis, 1997; Tzean et al., 1997). Isolation percentage of a particular species in soil was calculated using the formula: Isolation percentage = Number of positive soil samples for a particular species/ Total number of all samples × 100 (Abdullah and Mohamed, 2009). Pathogenicity bioassay: The pathogenicity trial was performed according to (Ali-Shtayeh et al., 2002). The tested fungal isolates were grown on PDA plates for 10 days. Sterile water (5 ml.) was powered on each plate containing fungal colony to obtain spore suspension, adjusted their concentrations at 1 x 108 conidia/ ml. Twenty adults of each of two aphid species (Hyalopterus pruni (Geoff.)) and Aphis pomi (DeGreer) were sprayed with 10 ml of spore suspension for few seconds for each isolate and then transferred into a sterile plates containing two pieces of moistened filter papers and two host plant leaves. Plates were sealed with Parafilm to maintain the humidity and then incubated in darkness at 25 oC. Infected dead insects were 21 Hasan, et al inspected and counted daily. Mortality percentage caused by each isolate was assessed after 2, 4 and 6 days. The experiment was conducted as a completely randomized design with four replicates for each isolate. Differences between the treatments were determined by ANOVA and Duncan test at P ≤ 0.05 with SAS software (SAS, 1999). RESULTS A total of 9 species assigned to 7 genera were recovered from 13 soil samples by the dilution method (Table 1). Penicillium glabrum was the most frequent species detected from all soil samples, followed by Aspergillus niger with 76.92% isolation rate .The two entomopathogenic fungi (Beauvaria bassiana and Isaria javanica) and Rhizopus stolonifer were each detected with 38.96% isolation rate. Other opportunistic fungi such as Alternaria alternate, A. flavus, Penicillium digitatum and Syncephalastratum racemosus were also isolated with isolation rates varying between 7.67% - 30.77%. The present study revealed that entomopathogenic and other opportunistic fungi are common inhabitant of soil at insect hibernation sites, however, their diversity is relatively low as indicated by the isolation of two entomopathogenic species and seven opportunistic fungi. Isaria javanica was isolated from Iraqi soil for the first time. The newly recorded species is described and illustrated. Phenotypical characterization of Isaria javanica (Frieder. & Bally) Samson & Hywel-Jones. Mycol.Res.109, 588 (2005). Fig.1(A-B). Colonies on PDA, growing slowly reached a radial of 4.6 mm in 14 days at 25 C, powdery to floccose, at first white becoming cream-coloured with age. Hyphae hyaline, septate, branched, smooth walled, 2-3um wide. Conidiophores erect, hyaline, simple or branched, up to 50 um long and 2-2.5um wide, forming verticillate branches with phialides in whorls of 2 to 3. Phialides 10-16 x 2-3 um, consisting of cylindrical basal portion tapering into a thin distinct neck. Conidia hyaline, smooth, one celled, fusiform, sometimes cylindrical, 5-6.5 x 2-2.3 um. Chlamydospores not observed. The pathogenicity test (Table 2) showed that B. bassaiana was the most virulent species causing 100% mortality to both aphid species (Hyalopterus pruni and Aphis pomi) after six days, followed by I. javanica (66.7% and 75.6% mortality) to both aphid species respectively. DISCUSSION In our study we have isolated surviving entomopathogenic and opportunistic fungi from diversely soil environments. This indicates that these fungi can be naturally found close to phytophagous insects host. Most fungi found in Iraq during this work have been reported from other parts of the world (Vanninen,1995; Meyling and Eilenberg, 2006). Regarding the entomopathogenic fungal species, B.bassiana was among the most frequently isolated fungi from soil at insect hibernation sites. This result is in agreement with several other studies, revealing that B. bassiana was encountered from a variety of agricultural and natural soils (Ali-Shtayeh et al.2002; Meyling and Eilenberg, 2006; Quesada- Movaga et al. 2007; Sun and Lin.2008; Sun et al.,2008). Moreover, the fungus seems has a wide distribution over the country and has been repeatedly isolated from different soils in Iraq 22 Occurrence of Entomopathogenic as well as from different insect hosts (Khalaf et al.1997, 1998; Assaf, 2007, 2009; Abdullah and Mohamed, 2009; Assaf et al.2011). Isaria javanica (Frieder.&Bally)Samson & Hywel-Jones (formerly known as Paecilomyces javanicus (Frider & Bally) A. H. S. Brown&G.Smith) and was originally described by Friederich & Bally (1923) as Spicaria javanica. The species is isolated from diverse soils at insect hibernation sites for the first time in Iraq. The description of our isolate is in agreement with Samson (1974), Tzean et al. (1997) and Shimazu and Takastuka (2010). Our isolate of I. javanica did not form synnemata, however, Samson (1974) described that P. javanicus occasionally produces a few synnemata which was not reported by other authors (Brown and Smith, 1957; Tzean et al.1997; Shimazu and Takatsuka, 2010). Performance of pathogenicity test for our isolate on two aphid species (H.pruni and A.pomi) caused 66.7% and 75.6% mortality respectively. Most reported host insects for I.javanica are members of either Lepidoptera or Coleoptera (Tzean et al. 1997; Chen et al.2007; Hu et al. 2007; Spacht et al., 2009; Shimazu and Tketsuko, 2010). The pathogenicity of the fungus was also proved on insects in Hemiptera (Scorselli et al., 2008) and in Hymenoptera (Hu et al. 2011). The species was also reported as an entomopathogenic fungal endophyte being isolated from peduncles of coffe plants (Vega et al. 2008). Several species in the genus Isaria (formerly Paecilomyces) such as I. farinosa (Holmsk.) Fr. and I. fumosorosea Wize, are well known insect pathogens and frequently isolated from soil (Ali-Shtayeh et al.2002; Meyling and Eilenberg, 2006; Sun and Liu,2008; Hu et al.2010). I. farinosa have been previously reported from Iraq as P. farinosus on Sunn pest and aphids (Assaf, 2007, 2009). Aspergillus flavus and A.niger isolated in the present study have previously been isolated as insect pathogens by several authors ((Sur et al., 1999; Abdullah et al.2001,2002; Sun and Liu, 2008; Abdullah and Mohamed, 2009 and Assaf et al., 2011). Several other fungal species including Penicillium glabrum, P. digitatum, Alternaria alternata, Syncephalastratum racemosum and Rhizopus stolonifer were detected with different isolation percentage. Though we considered these species as secondary colonizers, but these opportunistic fungi were proved their pathogenicity on different insect orders (Gunde-Cimerman et al., 1998; Abdullah et al.2002; Ali-shtayeh et al., 2002; Sun et al., 2008 and Abdullah and Mohamed, 2009). In conclusion, the present study provides the first report of I. javanica from Iraq as an entomopathogenic fungus, extending our knowledge of the occurrence and distribution of entomopathogenic fungi in Iraqi soil. LITERATURE CITED Abdullah S.K., Mohamed A.M. 2009. Occurrence of insect associated fungi in cultivated soil in Basrah, Iraq. Proceeding of the 1st scientific conference of Biological Sciences 22-23 April, (2009.), Mosul, Iraq. pp 222-227. Abdullah S.K., Hassan K.S., Mansour Z.F. 2001. Mycoflora associated with the subterranean termite Micocerotemes diversus in Basrah, Iraq. Iraqi J.Biol.1:109-116. Abdullah S. K., Hassan K. S., Mansour Z. F. 2002. 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Appl. Entomol. 102:213-215. Table 1: Isolation % of entomopathogenic and opportunistic fungi isolated from soil samples. Fungal species N˚ positive samples Isolation % Alternaria altenata 1 7.69 Aspergillus flavus 2 15.38 A. niger 10 76.92 Beauveria bassiana 5 38.46 Isaria javanica 5 38.46 Penicillium digitatum 4 30.77 P. glabrum 13 100.0 Rhizopus stolonifer 5 38.46 Syncepalastratum racemosum 1 7.69 Table 2: Pathogenicity trial of fungal isolates on H. pruni and A. pomi. Fungus species Insect species % Mortality 2 days 4 days 6 days Control H. pruni 0 e * 0 e 10 e A. pomi 5 de 10 de 15 de B. bassiania H. pruni 45 a 85 a 100 a A. pomi 31.58 ab 88.89 a 100 a P. javanicus H. pruni 20 bc 55 b 66.67 b A. pomi 26.32 bc 50 b 75.59 b A. nigur H. pruni 15 cd 25 c 27.78 cd A. pomi 5.26 de 22.22 cd 35.29 c Penicillium glabrum. H. pruni 15 cd 30 c 33.33 c A. pomi 21.05 bc 27.78 c 29.41 cd * Means followed by the same letters in each column are not significantly different (P = 0.05). 26 Occurrence of Entomopathogenic 27 Hasan, et al Bull. Iraq nat. Hist. Mus. (2012) 10 (1):19-27 تواجد الفطریات الحشریة واالنتھازیة فى ترب مواقع تشتیة الحشرات وتقیم القابلیة االمراضیة *سمیر خلف عبدهللا *لزكین حجى عساف *وزیر على حسن العراق - جامعة دھوك -لزراعھ والغابات كلیة ا -قسم وقایة النبات * العراق - جامعة زاخو -كلیة العلوم -قسم علوم الحیاة * الخالصة تم عزل الفطریات الممرضھ للحشرات واالنتھازیھ من عینات تربھ جمعت من اماكن تشتیة الحشرات فى بیئات مختلفھ فى كردستان العراق خالل الفتره تم عزل نوعین منى الفطریات . ٢٠٠٩كانون األول -من تشرین االول الحشریة Beauveria bassiana (Bals.) Vuill.and Isaria javanica (Friedrichs & Bally) Samson & Hywel-Jones. تم عزل . لكل فطر% ٣٨,٦التربة بأستخدام طریقة التخفیف وبتردد من :فطریات اخرى انتھازیة االمراضیة مثل Alternaria alternata, Aspergillus flavus, A.niger, Penicillium glabrum, P.digitatum, Rhizopus stolonifer and Syncephalastratum racemosum. على كال % ١٠٠كان االكثر ضراوة واحدث نسبة قتل B. bassiana الفطر نسبة قتل تراوحت مابین I. javanicaاظھر الفطر . النوعین من الحشرات تم عزل . على كال النوعین من الحشرات على التوالي% ٧٥,٥٩و% ٦٦,٦٧ تم وصف النوع المسجل مع . الول مرة في العراق I. javanicaوتسجیل النوع .التوضیح بالصور الفوتوغرافیة