Journal of Plant Protection Research ISSN 1427-4345 ORIGINAL ARTICLE Pathogenicity of endogenous isolate of Paramyrothecium (=Myrothecium) roridum (Tode) L. Lombard & Crous against the squash beetle Epilachna chrysomelina (F.) Feyroz Ramadan Hassan1*, Nacheervan Majeed Ghaffar2, Lazgeen Haji Assaf3, Samir Khalaf Abdullah4 1 Department of Plant Protection, College of Agricultural Engineering Sciences, University of Duhok, Kurdistan Region, Duhok, Iraq 2 Duhok Research Center, College of Veterinary Medicine, Duhok University, Kurdistan Region, Duhok, Iraq 3 Plant Protection, General Directorate of Agriculture-Duhok, Kurdistan Region, Duhok, Iraq 4 Department of Medical Laboratory Techniques, Al-Noor University College, Nineva, Iraq Vol. 61, No. 1: 110–116, 2021 Abstract DOI: 10.24425/jppr.2021.136271 The squash beetle Epilachna chrysomelina (F.) is an important insect pest which causes se- vere damage to cucurbit plants in Iraq. The aims of this study were to isolate and character- Received: September 14, 2020 ize an endogenous isolate of Myrothecium-like species from cucurbit plants and from soil Accepted: December 8, 2020 in order to evaluate its pathogenicity to squash beetle. Paramyrothecium roridum (Tode) L. Lombard & Crous was isolated, its phenotypic characteristics were identified and ITS *Corresponding address: rDNA sequence analysis was done. The pathogenicity ofP. roridum strain (MT019839) was [email protected] evaluated at a concentration of 107 conidia · ml–1) water against larvae and adults of E. chry­ somelina under laboratory conditions. The results revealed the pathogenicity of the isolate to larvae with variations between larvae instar responses. The highest mortality percentage was reported when the adults were placed in treated litter and it differed significantly from adults treated directly with the pathogen. Our results documented for the first time that P. roridum has potential as an insect pathogen. Keywords: Epilachna chrysomelina, Paramyrothecium roridum, pathogenicity, soil Introduction Paramyrothecium roridum (Tode) L. Lombard & Crous Paramyrothecium roridum produces several bioac- was introduced by Lombard et al. (2016) based on My­ tive secondary metabolites or toxins such as trichothe- rothecium roridum Tode as the type species. The spe- cens macrolides, including epiroridin E, mytoxin B cies is known to occupy different ecological niches. (Liu et al. 2016), myrothecin A, 8-alpha-hydroxyrori- It has been reported as a pathogen on different host din H (Soliman 2020), roridin E, verrucarin A (Bosio plants (Han et al. 2014; Haudenshield et al. 2018; Matic et al. 2017), myrothecines, hydroxymytoxin B and se- et al. 2019; Pappachan et al. 2019; Soliman 2020), as veral other cyclotrichothecan derivatives (Shen et al. a seed-borne pathogen (Bharath et al. 2006; Abdullah 2019). These compounds show cytotoxic activities on and Al-Mosawi 2010), as an endophyte (Amitha et al. human cells (Shen et al. 2019) and tumor cell lines 2014; Liu et al. 2016; Shen et al. 2019), or as a soil and (Liu et al. 2016). Some metabolites also have clear an- dead plant substrate inhabitant (Domsch et al. 2007; tibiotic and antifungal effects against a wide range of Lee et al. 2008; Abdullah et al. 2010; Kwon et al. 2014; bacteria and fungi (Liu et al. 2006). Moreover, due Lombarad et al. 2016). to its phytotoxic effects. P. roridum is considered to Feyroz Ramadan Hassan et al.: Pathogenicity of endogenous isolate of Paramyrothecium … 111 be a promising agent for biological weed control (Lee cultivated with different cucurbit plants such as cu- et al. 2008). cumber, squash, snake cucumber, melon, watermelon Several studies showed that P. roridum isolates are and pumpkin from seven villages in the Amadia dis- capable of producing various extracellular hydrolytic trict. The soil samples, about 500 g each, were taken enzymes such as cellulases (Cx) and polygalacturonase randomly from a depth of 0–10 cm (standard depth (PG) as revealed by (Okunowo et al. 2010; Talukdar of sampling for fungi) with a trowel after removing and Dantre 2014; Soliman 2020). Production of tox- litter or weed plants. They were then placed in plas- ins and extracellular hydrolytic enzymes contributed tic bags, labeled and transferred to the laboratory significantly to its pathogenicity (Okunowoet al. 2010; (25 ± 2°C). Nine soil samples/cucurbit plant/village Soliman 2020). were taken. Before using, the samples were thoroughly The insecticidal activity of M. roridum has never mixed and passed through a fine mesh sieve to break been reported on insects except by Mou (1975) who up soil lumps and separate litter remnant. Paramy­ found that some insects, such as Macrotermes barneyi rothecium species were isolated with direct soil plating and Dendrolimus punctatus, are parasitized by taxa (Warcup 1960). The plates were incubated at 25°C for which are morphologically similar to M. roridum. 7 days, then checked for the occurrence of Paramyro­ In Iraq, in previous studies, Paramyrothecium thecium spp. and other opportunistic fungi from the soil (=Myrothecium) roridum (Tode) L. Lombard & Crous using oat amended with cetyl trimethyl ammonium has been isolated from different sources including bromide CTAB (0.6) as selective media. Pure cultures soil around Basrah (Ismail and Abdullah 1977), soil from the obtained growing colonies were transferred from date palm plantations (Abdullah and Zora 1993), to fresh appropriate potato dextrose agar (PDA) me- surface sediments of the Shatt Al-Arab river (Abdul- dia for identification depending on their morphologi- lah and Abbas 2008), sunflower seeds (Abdullah and cal cha racteristics and reproductive structures with Al-Mosawi 2010) and soil from grapevine nurseries the aid of several taxonomic references (Tulloch 1972; at Duhok (Abdullah and Saadullah 2013). However, Lombard et al. 2016). To isolate entophytic Paramyro­ the isolates were only identified according to their thecium, cucurbit plants were collected and transferred morphological characteristics. The squash beetle, Epi­ to a laboratory. For each plant, healthy tissues (root, lachna chrysomelina (F.) is a common pest of cucurbit stem and leaves) were surface-sterilized according to plants in Iraq (Abdul-Rassoul 1976). Both larvae and Arnold (2007) and Macia-Vicente et al. (2008), plated adults cause severe damage to the leaves which re- on selective medium (30 g oat, 0.6 CTAB, 15 g agar, duces the vegetative production of the host plant and 0.25 g chloramphenicol, 1 l distilled water) and incu- leads to yield reduction, growth discontinuation, fruit bated at 25°C for 2 weeks to record the occurrence of degradation and even complete dryness of the plants Paramyrothecium spp. and other opportunistic fungi. (Awadalla et al. 2011). In Iraq, the control of this pest Pure cultures from the obtained growing colonies were has focused on using insecticides and no biological transferred to fresh appropriate PDA medium for iden- agents have been reported on E. chrysomelina except tification. Colony morphology, growth rate and mor- for the study of Hassan et al. (2019). They evaluated phological characteristics of isolates were recorded. the pathogenicity of two isolates of B. bassiana against various stages of E. chrysomelina under laboratory and Morphological observations field conditions to develop application strategies suit- able for future use in biological control. To produce monosporic cultures, a conidial suspen- In this study we aimed to identify P. roridum isolat- sion of 1 × 10 conidia · ml–1 was prepared from fungal ed from cucurbit plants and the soil by using morpho- cultures grown on PDA plates for 2 weeks. A single logical and molecular analysis with ITS-rDNA region colony reproduced from single conidia was transferred amplification and sequencing. Then we evaluated its to a new PDA dish and incubated at 25°C. Microscopic pathogenicity to squash beetle E. chrysomelina. measurements of conidia were taken from slide-cul- tures produced by inoculation of a small amount of mycelium on a drop of methylene blue stain and cov- Materials and Methods ered with a cover slip. Measurements were performed with graticule lens. Isolation Genomic DNA extraction, PCR Cucurbit plants and soil were collected from Ama- and sequencing dia district (N 37.0917°, E43.4877°, 1122 m above sea level), Duhok province, Kurdistan region, Iraq. The To identify the Paramyrothecium isolates at a mole- soil samples were collected from agricultural soils cular level, they were grown on potato dextrose broth 112 Journal of Plant Protection Research 61 (1), 2021 for 7 days at 25°C. The mycelia samples were pelleted and used. Tween 80 at a concentration of 0.02% was from liquid culture by filtering and then frozen and added to the suspension. Conidia concentration was stored at –20°C. For polymerase chain reaction (PCR) measured using a hemocytometer slide. amplification, genomic DNA was extracted from the Replicates were determined (10 adults/replicate) in prepared mycelium mat according to the commercial a small plastic container (20 × 10 × 10 cm) lined with animal and fungi DNA preparation kit protocol (Jena moistened filter paper, supplied with fresh and clean Bioscience, Germany) (Hassan 2019). A set of Primers pieces of pumpkin leaves and fruit when required. The ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) and ITS5 replicates were sprayed using a new sprayer (50 ml ca- (5’-GGAAGTAAAAGTCGTAACAAGG-3’) was used pacity). The application was repeated twice. to amplify an internal transcribed spacer