ABSTRACT Five moist soil zones in temperate forests of Pakistan were investigated for the bioactive potential of actinomycetes. The identiication of the isolates was based on their cultural and morphological characteristics among which 60 isolates were screened and recognised out of 208 isolates. The isolates identiication falls under three genera including Actinomyces, Streptomyces and Nocardia spp. each with the total number of 31, 17 and 12 isolates identiied respectively. The identiied isolates were further screened for bioactive potential among which 15 isolates produced bioactive substances against one or more indicator strains of gram positive and gram negative bacteria and fungi. The results clearly demonstrated that temperate forest ecosystems are providing a key habitat to bioactive actinomycetes, which have an important medical, scientiic and economic signiicance globally in general and in particular for developing countries like Pakistan. Actinomycetes screening for bioactive potential isolated from the moist forest soils of Pakistan Imran Ullah 1 , Masood Arshad 1 , Muhammad Jamshed Iqbal Chuadhry 1 , Uzma Noureen 1 , Waqar Azeem Jadoon 2 , Muhammad Ayub Jadoon 3 1 WWF - Pakistan, House No. 451, Street No. 2, Sector F-11/1, Islamabad, Pakistan 2 Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan 3 Department of Microbiology, Hazara University, Mansehra, Pakistan Corresponding author: Imran Ullah (Email: imranhu1@yahoo.com) ARTICLE INFO Keywords Bioactive potential Temperate forest Moist soils Actinomyces Streptomyces Nocardia Introduction Actinomycetes are gram-positive bacteria that form ilamentous mycelia with high G+C (guanine +cytosine) content and are widely distributed in a variety of natural and manmade environments, particularly constituting a signiicant component of the microbial population in temperate forest soils (Debananda et al., 2009; Lam, 2006 and Ndonde & Semu, 2000 and Watve et al., 2001). About 100 genera of actinomycetes exist in natural habitats including diverse forest zones (Yokota, 1997). Some genera such as Streptomyces and Actinoadura are widely distributed, which can be isolated from different temperate soil habitats (Williams et al., 1989). Among the gram-positive bacteria, Actinomycetes exhibit the greatest morphological differentiation with branching hyphae and specialised spore-bearing structures (Kim & Garson, 2005; Prescott et al., 1993). These bacteria are globally signiicant and have been extensively studied (Cragg & Newman, 2005; Bull et al., 2000 and Debananda et al., 2009), due to their ability to produce novel antibiotics (Goodfellow et al., 1989; Williams et al., 1983; Crandall & Hamil, 1986; Williams et al., 1989 and Korn & Kutzner, 1992). Of one thousand different antibiotics known today, more than 70 % are produced by Actinomycetes (Bull & Stach, 2005; Edward, 1980; Imada & Okami, 1998; Kim & Garson, 2005). In addition to antibacterial activity, actinomycetes also produce commercially important bioactive compounds such as avermectin (Prescott et al., 1993) and other secondary metabolites with biological activities (Blunt & Prinsep, 2006 and Debananda et al., 2009) hence actinomycetes strains have many gene clusters involving the biosynthesis of melanin, carotenoid, siderophore, polyketide and peptide compounds (Omura et al., 2001). To our knowledge, actinomycetes isolation from temperate forest soils in Pakistan have not been undertaken by researchers and this geographic region may have important bioactive actinomycetes traits, which could be of medical and economic beneits to Pakistan. The current study was conducted to isolate potential soil actinomycetes strains from ive diverse geographic regions, which fall in the temperate forests of Pakistan. The isolates were further screened for bioactive potential against selected strains of gram positive and gram negative bacteria and fungi. Material and Methods Study Area Five temperate forest zones from diverse geographic locations: Miadam Forest Swat (MFS), Kalam Forest Swat (KFS) and Osherai Dara Forest Dir (OFD), Ayubia Forest Hazara (AFH), Nathiagali Forest Hazara (NFH) of Malakand and Hazara Divisions in north western Pakistan, respectively (Fig. 1) were selected for soil sampling and subsequent isolation and screening of bioactive actinomycetes. These temperate forests zones having suitable rich soil conditions (~40 to 60 % volumetric water content and 5 to 8 % organic carbon on dry soil mass basis), acting as a growing media for bioactive actinomycetes, and these conditions provided the preliminary basis for selection of the study area for soil sampling. Data Collection Soil Sample collection and pre-treatment The soil samples were collected during May, 2009 to February, Figure 1: Soil sampling sites in temperate forest zones of north-western Pakistan 10 Rec. Zool. Surv. Pakistan 21: 10-13 (2012)