Pak. J. Bot., 50(4): 1621-1628, 2018. ROLE OF ENDOPHYTIC PENICILLIUM SPECIES IN SUPPRESSING THE ROOT ROTTING FUNGI OF SUNFLOWER FAIZAH UROOJ1, HAFIZA FARHAT1, SYED ABID ALI2, MARIYAM AHMED1, VIQAR SULTANA3, ZAFAR IQBAL SHAMS4, JEHAN ARA5 AND SYED EHTESHAMUL-HAQUE1* 1 Agricultural Biotechnology & Phytopathology Laboratory, Department of Botany, University of Karachi, Karachi 75270, Pakistan 2 HEJ Research Institute of Chemistry, (ICCBS), University of Karachi, Karachi-75270, Pakistan 3 Department of Biochemistry, University of Karachi, Karachi-75270, Pakistan 4 Institute of Environmental Studies, University of Karachi, Karachi-75270, Pakistan 5 Department of Food Science & Technology, University of Karachi, Karachi-75270, Pakistan * Corresponding author’s email: sehaq@uok.edu.pk Abstract Micro-fungi have been the source of novel and pharmacologically active compounds over decades. Among them, genus Penicillium has been recognized as a rich source of bioactive metabolites. In this study, out of 80 plant samples, endophytic Penicillium species were isolated from 14 samples (root, stem and leaves) and identified as P. asperum, P. citrinum, P. duclauxi, P. javanicum, P. lividum, P. nigricans, P. decumbens, P. purpurogenum (3 isolates), P. lilacinum, P. restrictum, P. rugulosum and P. thomii. In dual culture plate assay all the fourteen isolates of Penicillium inhibited all four test fungi Fusarium oxysporum, F. solani, Rhizoctonia solani and Macrophomina phaseolina by producing zone of inhibition. Cell free culture filtrate also showed strong antifungal activity at higher dosages. Most of the isolates were significantly suppressed root rotting fungi and improved growth of sunflower in screen house experiments when applied alone or in soil amended with neem (Azadirachta indica A.Juss) cake. Key words: Endophytes, Penicillium, Antifungal, Root rotting fungi, Sunflower. Introduction Interactions of plants with microorganisms exist as a composite network; some of them may be beneficial, while others are harmful. But the beneficial microorganisms are considerably largest and still broadly unexplored. Some of the beneficial microorganisms, which colonize healthy plant tissue without causing any apparent symptoms of disease, called endophytes (Gherbawy & Gashgari, 2014; Shafique et al., 2015; Korejo et al., 2017). They are increasingly gaining scientific and commercial interest because of their potential to improve plant growth through and suppression of plant diseases (Afzal et al., 2013; Khan & Lee, 2013). Endophytes produce several compounds that promote growth of plants and help them adapt better to the environment (Rashid et al., 2012). Endophytes play crucial role in phyto-stimulation, pigment and enzyme production, antimicrobial activity, bioactive and novel compound production, nutrient cycling and bioremediation (Nair & Padmavathy, 2014). Antifungal and antibacterial activities of plant endophytic fungi have been reported by several researchers (Bhardwaj et al., 2015; Gherbawy & Gashgari, 2014). Among the endophytic fungi genus Penicillium has been recognized as a rich source of bioactive metabolites (Fill et al., 2007). Penicillium is one of the largest and most important genera of microscopic fungi, with over 400 described species, distributed worldwide (Visagie et al., 2014). Penicillium species are generally considered as soil inhabitant or as contaminant of food, fruits, fibers and other starchy materials, which were found to play important role in plants against stress (Ali et al., 2011; Khan & Lee, 2013). Penicillium species produce a range of medicinally important metabolites including antimicrobial (Lucas et al., 2007), antifungal (Nicoletti et al., 2007), anti-cancer and insecticidal (Singh, 2003; Stierle et al., 2006) and nematicidal (Qureshi et al., 2012). In our previous study, we have reported antimicrobial activity of endophytic Penicillium species, isolated from Salvadora species (Korejo et al., 2014). The present report describes the isolation and identification of endophytic Penicillium species from wild and cultivated plants and their biocontrol potential against root rotting fungi infecting sunflower. Materials and Methods Collection of plant samples and site: In this study, 80 healthy plant samples (with hypothesis that they harbor unique microbial community) belonging to 29 plant species viz., Abelmoschus esculantus (L.) Moench, Achyranthus aspera L., Allium cepa L., Amaranthus sp., Arachis hypogea L., Atriplex stocksii Boiss., Azadirachta indica A.Juss., Brassica campestris L., Capsicum annuum L., Carica papaya L., Chenopodium sp., Citrullus lanatus (Thunb.) Mansf., Corchorus tridens L., Cucurbita pepo L., Cyamopsis tetragonoloba (L.) Taub., Euphorbia hirta L., Helianthus annuus L., Lactuca sativa L., Luffa aegyptiaca Mill., Lycopersicon esculentum Mill., Momordica charantia L., Pennisetum glaucum (L.) R.Br., Solanum melongena L., Sorghum bicolor (L.) Moench, Spinacea oleracea L., Tribulus terristeris L., Trigonellafoenum- graecum L., Triticum aestivum L. and Zea mays L. were collected from agricultural fields of Malir, Karachi (Memon Goth, Kathor) and Karachi University. 1622 Karachi is located between 24o 45’ N to 25o 37’ N and 66o 42’ E 67o 34 E along the shoreline of the Arabian Sea. The area is classified as arid hot desert. It is naturally a shrub land. It experiences low average precipitation (25 cm per annum). Its seasonal temperature fluctuates between 13oC to 36oC and rarely falls below 9oC during winters and elevates above 36oC during summers. Soil in Campus Area is sandy loam and mostly alkaline in nature (electrical conductivity 0.80-2.80 mS cm-1) with maximum water holding capacity is 23.50–41.00% (Rab et al., 2016). Soil characteristics in Memon Goth was similar to Campus, whereas in Kathor, soil contains higher percentage of sands. Isolation of endophytic Penicillium was made within 24 hours. Isolation and identification of endophytic Penicillium: One gram of plant samples (roots, stems and leaves) was separately washed under tap water, sterilized with 1% bleach for 3 minutes, then with 70% alcohol for 3 minutes and finally washed with distilled water. Each sample was chopped in sterilized grinder with 50mL sterilized water and dilutions of each sample were made up to 1:10 4 and 0.1mL suspension from final dilution was transferred onto a Petri-dish containing Potato Dextrose Agar (PDA) supplemented with penicillin (100,000 units/liter) and streptomycin (0.2gm/liter). The plates were incubated at 28°C for 5 days and fungi were identified with reference to Barnett & Hunter (1998), Raper & Thom (1949) and Visagie et al., (2014). In vitro dual culture plate assay for determining the antifungal activity of Penicillium species: Antifungal activity of Penicillium species was made against four common root rotting fungi viz., Macrophomina phaseolina, Rhizoctonia solani, Fusarium solani and F. oxysporum. A 5mm agar disc of test Penicillium was inoculated on one side of 90mm Petri-dish containing Czapek’s Dox Agar, pH7.2. On the other side of same Petri-dish, a 5mm disc of test pathogen was inoculated and incubated at 28°C for 5 days. Zone of inhibition was measured, averaged and expressed in mm (Korejo et al., 2014). There were four replicates of each test and repeated twice. Preparation of culture filtrates and antifungal activity: Test Penicillium species were grown in 250mL conical flask containing 100mL Czapek’s Dox broth. Each flask was inoculated with 5mm disc of test Penicillium, cut from the margin of vigorously growing culture. The flasks were incubated for 15 days at room temperature (25-30°C). After 15 days, test fungi were filtered and culture filtrates were collected in sterile flasks. The culture filtrates were then exposed to chloroform vapors to kill propagules of Penicillium, if any. To examine the antifungal activity of secondary metabolites of Penicillium species thick sterile filter paper discs were loaded with sterile culture filtrate of each Penicillium species at 20, 40 and 60µl/disc and dried. These discs were placed at different places of plates containing Czapek’s Dox Agar. In the center of Petri dishes a 5mm disc of test fungus was inoculated. Discs loaded with sterile broth of Czapek’s Dox broth were served as FAIZAH UROOJ ET AL., control, whereas carbendazim at 20µg/disc were served as positive control. Petri dishes were incubated at 30°C for 5-7 days and distance between test fungus and disc was considered as zone of inhibition (Qureshi, 2003). Effect of endophytic Penicillium species on root rotting fungi on sunflower: The experiment was carried out in earthen pots (15cm diam.) in screen house, where neem (Azadirachta indica A. Juss) cake (Neemex powder), purchased from Sigma Energy (pvt) Ltd, Karachi, was mixed with sandy loam soil (pH8.0) at 1% w/w and transferred to each pot at 1 Kg per pot. The soil was naturally infested with root rotting fungi Macrophomina phaseolina (2-8 sclerotia g-1 of soil) as determined by wet sieving and dilution plating (Sheikh & Ghaffar, 1975), 310% colonization of Rhizoctonia solani on sorghum seeds used as baits (Wilhelm, 1955) and 3000 cfu g-1 of soil of a mixed population of Fusarium oxysporum and F. solani as determined by soil dilution technique (Nash & Snyder, 1962). The pots were watered for 7 days to allow complete decomposition of organic matter. Sunflower (Helianthus annuus) seeds were sown in each pot at 6 seeds per pot and 25mL aqueous suspension of Penicillium species (8x107 cfu/mL) grown in Potato Dextrose broth was drenched onto each pot. After germination four seedlings were kept in each pot and excess were removed. In another set, Penicillium was inoculated in un-amended soil for comparison. Plants grown in un-amended or un-inoculated soil are served as control, while carbendazim (25mL of 200ppm) are served as positive control. Observations were recorded after 45 days. To assess the efficacy of soil amendment with neem cake and Penicillium, plants were uprooted and roots were washed thoroughly with sterilized water and the causal fungi were isolated as described by Mansoor et al., (2007). Fungi, which were emerged from root pieces, were identified and infection percentage were calculated. Data on plant growth was also recorded. The experiment was conducted in March 2016 and repeated in March 2017 with similar conditions to confirm the results. Results Isolation of endophytic Penicillium species and growth inhibition of root rotting fungi by Penicillium species in dual culture plate assay: Out of 80 plant samples (roots, stems and leaves) examined, endophytic Penicillium were isolated from 14 samples, belonging to 12 species (Table 1). Isolates of Penicillium were identified as P. asperum, P. citrinum, Penicillium species, P. duclauxi, P. javanicum, P. lividum, P. nigricans, P. decumbens, P. purpurogenum (3 isolates), P. lilacinum, P. restrictum, P. rugulosum and P. thomii (Table 1). All isolates of Penicillium were tested against 4 root rotting fungi M. phaseolina, R. solani, F. solani and F. oxysporum in vitro. In dual culture plate assay, all of the subjected 14 isolates showed antifungal activity by producing zone of inhibition (Table 1). Penicillium duclauxi and P. lividum produced large sized zone (more than 10mm) against all the four test fungi compared to other isolates. 1623 BIOCONTROL POTENTIAL OF ENDOPHYTIC PENICILLIUM SPECIES Table 1. Growth inhibition of Macrophomina phaseolina, Rhizoctonia solani, Fusarium solani and F. oxysporum in dual culture plate assay by the endophytic Penicillium species isolated from different wild and cultivated plants. Host name Plant part M. phaseolina R. solani F. solani F. oxysporum Fungus # Penicillium spp. EPSMR1 P. citrinum Solanum melongena L. (Solanaceae) Root 4 4 20 20 EPSMS2 P. lilacinum Solanum melongena L. (Solanaceae) Stem 6 8 11 14 EPSML3 P. purpurogenum Solanum melongena L. (Solanaceae) Leaf 6 5 25 17 EPSLR4 P. nigricans Lycopersicon esculentum Mill., (Solanaceae) Root 5 25 16 21 EPAAR5 P. rugulosum Achyranthus aspera L. (Amaranthaceae) Root 3 12 11 20 EPAIR6 P. decumbens Azadirachta indica A.Juss (Meliaceae) Root 5 25 13 20 EPEHS7 P. purpurogenum Euhorbia hirta L. (Euphorbiaceae) Stem 6 5 25 17 EPCTS8 P. restrictum Chorchorus tridens L. (Malvaceae) Stem 2 2 5 5 EPASS9 P. duclauxi Atriplex stocksii (Amaranthaceae) Stem 18 13 11 14 5 Zone of inhibition (mm) EPHAL10 P. asperum Helianthus annuus L. (Asteraceae) Leaf 2 2 5 EPAER11 Abelmoschus esculentus L. (Malvaceae) Root 5 8 5 6 EPMCL12 P. lividum Momordica charantia L. (Cucurbitaceae) Leaf 18 13 11 14 EPSLR13 Lycopersicon esculentum Mill., (Solanaceae) Root 5 24 17 22 Root 5 3 21 12 P. thomii P. javanicum EPAER14 P. purpurogenum Abelmoschus esculentus L. (Malvaceae) Growth inhibition of root rotting fungi by the cell-free culture filtrates of endophytic Penicillium species: Cellfree culture filtrate of Penicillium caused growth suppression of root rotting fungi viz; M. phaseolina, R. solani, F. solani and F. oxysporum In vitro (Table 2). Macrophomina phaseolina was inhibited by culture filtrates of P. lilacinum, P. nigricans and P. thomiiat 60µl/disc by producing maximum zone of 20 mm. P. lilacinum, P. nigricans and P. thomii also showed zone of inhibition of 15mm at 20µl/disc and 17mm at 40µl/disc. Rhizoctonia solani was inhibited by producing zone of 14mm at 60µl/disc from culture filtrates of P. lilacinum, P. purpurogenum (EPSML3), P. purpurogenum (EPEHS7), P. asperum and P. purpurogenum (EPAER14). P. nigricans and P. thomii produced zone of inhibition of 17mm at 60µl/disc against F. solani. Penicillium decumbens, P. citrinum, P. purpurogenum (EPSML3), P. regulosum, P. purpurogenum (EPEHS7), P. duclauxi, P. asperum, P. thomii, P. javanicum and P. purpurogenum (EPAER14) produced zone of inhibition ranging from 1214mm at 60µl/disc against F. oxysporum (Table 2). Suppression of root rotting fungi of sunflower by the endophytic Penicillium species in screen house experiment (2016): Plants that were grown in soil amended with neem (Azadirachta indica) cake, generally showed less infection of root rotting fungi compared to plants, which are grown in natural soil (un-amended soil). Most of the plants inoculated with endophytic Penicillium species showed less infection of root rotting fungi as compared to untreated control. Plants that were grown in pots received endophytic P. regulosum in natural soil and also in amended soil with neem cake showed no infection of F. oxysporum (Table 3). Whereas, P. decumbens, P. nigricans, P. regulosum, P. citrinum, P. purpurogenum (EPSML3), P. duclauxi, P. thomii, P. javanicum and P. asperum in amended soil with neem cake also showed no infection of F. oxysporum. Combined effect of isolates P. decumbens, P. nigricans, P. citrinum, P. lilacinum, P. purpurogenum (EPSML3), P. duclauxi, P. lividum, P. purpurogenum (EPEHS7), P. restrictum, P. thomii, P. purpurogenum (EPAER14), P. javanicum with neem cake showed no infection on F. solani. P. decumbens, P. nigricans, P. regulosum and P. javanicum also showed no infection of F. solani when used alone. P. lividum alone showed no infection of M. phaseolina on sunflower roots. Combined effect of P. decumbens, P. nigricans, P. regulosum, P. thomii and P. javanicum with Neem cake showed significant reduction on infection of M. phaseolina. Application of P. decumbens, P. nigricans, P. citrinum, P. lividum, P. purpurogenum (EPEHS7), P. purpurogenum (EPAER14) and P. javanicum showed no infection of R. solani. P. decumbens, P. regulosum, P. citrinum, P. lilacinum, P. purpurogenum (EPSML3), P. duclauxi, P. purpurogenum (EPEHS7), P. restrictum, P. purpurogenum (EPAER14), P. javanicum with neem cake showed no infection of R. solani. While P. nigricans, P. lividum, P. thomii and P. asperum significantly suppressed the R. solani infection when applied in neem cake amended soil (Table 3). Greater plant height was produced by P. purpurogenum (EPEHS7), P. restrictum, P. purpurogenum (EPAER14) and P. asperum when applied in neem cake amended soil. However, the effect of P. restrictum and P. asperum with neem cake were significant on fresh shoot weight (Table 4). Penicillium nigricans, P. thomii and P. javanicum alone showed significant result on root length and root weight whereas, P. decumbens and P. duclauxi with neem cake showed greater root length (Table 4). Suppression of root rotting fungi of sunflower by the endophytic Penicillium species in screen house experiment (2017): In the experiment repeated in 2017 generally showed less infection of root rotting fungi in plants, which were grown in soil amended with neem cake compared to plant that were grown in natural soil (un-amended soil). Plants, which were grown in pots, received endophytic Penicillium isolates that caused significant reduction of F. oxysporum except P. purpurogenum (EPSML3) and P. lividum (Table 5). Whereas pots received endophytic P. citrinum, P. purpurogenum (EPSML3), P. nigricans, P. regulosum, P. decumbens, P. duclauxi, P. thomii, P. javanicum showed complete suppression of F. oxysporum in neem cake amended soil. 1624 FAIZAH UROOJ ET AL., Table 2. In vitro growth inhibition of Macrophomina phaseolina, Rhizoctonia solani, Fusarium solani and F. oxysporum by culture filtrates of endophytic Penicillium species isolated from wild and cultivated plant species. M. phaseolina R. solani F. solani F. oxysporum Fungus No. Penicillium spp. Zone of inhibition (mm) Control 0 0 0 0 +ve control (Carbendazim 20µg/disc) 8 5 9 7 EPSMR1 P. citrinum 20 µl/disc 8 8 8 10 40 µl/disc 8 10 10 10 60 µl/disc 16 12 10 12 EPSMS2 P. lilacinum 20 µl/disc 15 10 10 5 40 µl/disc 17 10 12 5 60 µl/disc 20 14 12 8 EPSML3 P. purpurogenum 20 µl/disc 12 8 10 8 40 µl/disc 14 8 12 8 60 µl/disc 14 14 14 12 EPSLR4 P. nigricans 20 µl/disc 15 0 11 8 40 µl/disc 17 4 15 9 60 µl/disc 20 8 17 12 EPAAR5 P. rugulosum 20 µl/disc 11 6 8 9 40 µl/disc 16 10 8 12 60 µl/disc 16 12 12 12 EPAIR6 P. decumbens 20 µl/disc 12 5 14 12 40 µl/disc 14 8 14 14 60 µl/disc 14 8 14 14 EPEHS7 P. purpurogenum 20 µl/disc 12 8 10 8 40 µl/disc 14 8 12 8 60 µl/disc 14 14 14 12 EPCTS8 P. restrictum 20 µl/disc 8 0 8 8 40 µl/disc 10 5 8 9 60 µl/disc 11 7 12 11 EPASS9 P. duclauxi 20 µl/disc 12 0 12 10 40 µl/disc 16 6 14 10 60 µl/disc 16 8 14 12 EPHAL10 P. asperum 20 µl/disc 10 8 12 10 40 µl/disc 12 10 16 12 60 µl/disc 12 14 16 12 EPAER11 P. thomii 20 µl/disc 15 0 11 8 40 µl/disc 17 4 15 9 60 µl/disc 20 8 17 12 EPMCL12 P. lividum 20 µl/disc 12 8 10 9 40 µl/disc 12 8 12 11 60 µl/disc 14 12 13 11 EPSLR13 P. javanicum 20 µl/disc 10 0 8 8 40 µl/disc 12 5 9 8 60 µl/disc 14 8 10 12 EPAER14 P. purpurogenum 20 µl/disc 12 8 10 8 40 µl/disc 14 8 12 8 60 µl/disc 14 14 14 12 BIOCONTROL POTENTIAL OF ENDOPHYTIC PENICILLIUM SPECIES 1625 Table 3. Effect of endophytic Penicillium and neem cake on the infection of Fusarium solani, F.oxysporum, Rhizoctonia solani and Macrophomina phaseolina on sunflower roots in screen house experiment (2016). Infection % Treatments F. oxysporum F. solani M. phaseolina R. solani Code # NS AS NS AS NS AS NS AS Control -50 18.7 75 25 75 50 18.7 12.5 Carbendazim -25 0 31.2 6.2 12.5 25 12.5 0 P. decumbens EPAIR6 18.7 0 0 0 25 18.7 0 0 P. nigricans EPSLR4 6.2 0 0 0 37.5 18.7 0 6.2 P. regulosum EPAAR5 0 0 0 18.7 6.2 18.7 6.2 0 P. citrinum EPSMR1 37.5 0 25 0 12.5 25 0 0 P. lilacinum EPSMS2 25 6.2 18.7 0 6.2 50 6.2 0 P. purpurogenum EPSML3 50 0 12.5 0 6.2 25 6.2 0 P. duclauxi EPASS9 50 0 6.2 0 31.2 31.2 6.2 0 P. lividum EPMCL12 50 6.2 50 0 0 50 0 6.2 P. purpurogenum EPEHS7 37.5 18.7 37.5 0 50 31.2 0 0 P. restrictum EPCTS8 50 6.2 6.2 0 12.5 43.7 6.2 0 P. thomii EPAER11 6.2 0 6.2 0 37.5 18.7 6.2 6.2 P. purpurogenum EPAER14 37.5 18.7 37.5 0 50 31.2 0 0 P. javanicum EPSLR13 6.2 0 0 0 37.5 18.7 0 0 P. asperum EPHAL10 12.5 0 25 18.7 37.5 31.2 6.2 6.2 LSD0.05 Treatment=4.651 Pathogen=2.322 Soil Type=1.643 1. Mean values in the column showing difference greater than LSD value are significantly different at p<0.05 2. Mean values in the row showing difference greater than LSD value are significantly different at p<0.05 3. Mean values in the NS and AS column showing difference greater than LSD value are significantly different at p<0.05 NS= Natural Soil; AS=Amended Soil Table 4. Effect of endophytic Penicillium spp. and neem cake on the growth of sunflower in green house experiment (2016). Treatments Shoot length (cm) Shoot weight (g) Root length (cm) Root weight (g) Code # NS AS NS AS NS AS NS AS Control -22.7 39.9 2.53 5.35 6.4 11.6 0.64 0.67 Carbendazim -25.8 41.8 2.21 4.51 7.4 12.8 0.71 0.62 P. decumbens EPAIR6 25.4 44.8 2.43 5.12 11.0 14.0 0.77 0.78 P. nigricans EPSLR4 28.2 44.0 2.77 5.27 12.2 12.1 1.00 0.64 P. regulosum EPAAR5 25.2 44.0 2.5 4.75 8.6 12.8 0.78 0.62 P. citrinum EPSMR1 25.9 46.8 2.18 5.1 9.4 8.6 0.72 0.80 P. lilacinum EPSMS2 22.6 45.8 2.05 5.39 6.3 5.5 0.66 0.57 P. purpurogenum EPSML3 25.2 40.8 2.15 4.71 9.3 6.8 0.84 0.64 P. duclauxi EPASS9 25.4 44.8 2.43 5.12 11.0 14.0 0.77 0.78 P. lividum EPMCL12 22.6 45.8 2.05 5.39 6.3 5.5 0.66 0.57 P. purpurogenum EPEHS7 23.4 49.3 1.53 5.73 8.8 7.2 0.58 0.74 P. restrictum EPCTS8 26.1 49.1 2.14 6.78 9.1 7.5 0.69 0.86 P. thomii EPAER11 28.2 44 2.77 5.27 12.2 12.1 1.00 0.64 P. purpurogenum EPAER14 23.4 49.3 1.53 5.73 8.8 7.2 0.58 0.74 P. javanicum EPSLR13 28.2 44 2.77 5.27 12.2 12.1 1.00 0.64 P. asperum EPHAL10 26.2 49.1 2.14 6.78 9.1 7.5 0.69 0.86 LSD0.05 5.11 7.81 0.791 1.821 2.51 2.81 0.1951 0.31 1. Mean values in the column showing difference greater than LSD value are significantly different at p<0.05 NS= Natural Soil; AS= Amended Soil Combined effect of P. nigricans, P. citrinum, P. lilacinum, P. lividum, P. restrictum, P. thomii, P. javanicum and neem cake showed no infection of F. solani. P. decumbens, P. nigricans and P. javanicum that also showed complete suppression of infection of F. solani. Plant that received P. lividuma alone showed no infection of M. phaseolina on sunflower roots. Combined effect of all treatments with neem cake showed significant reduction in infection of M. phaseolina. Application of P. decumbens, P. citrinum, P. lividum, P. purpurogenum (EPEHS7), and P. regulosum showed no infection of R. solani. P. decumbens, P. nigricans, P. citrinum, P. purpurogenum (EPSML3), P. duclauxi, P. purpurogenum (EPEHS7), P. restrictum, P. purpurogenum (EPAER14) and P. javanicum with neem cake showed complete suppression of R. solani (Table 5). Furthermore, plants which were grown in soil amended with neem cake generally showed greater height compared to plant grown in natural soil (unamended soil). Most of the plants inoculated with endophytic Penicillium species showed larger shoot length compared to untreated control. Greater plant height was produced by P. lilacinum when applied in neem cake amended soil (Table 6). 1626 FAIZAH UROOJ ET AL., Table 5. Effect of endophytic Penicillium and neem cake on the infection of Fusarium solani, F. oxysporum, Rhizoctonia solani and Macrophomina phaseolinaon sunflower roots in green house experiment (2017). Infection% Treatments Code # F. oxysporum F. solani M. phaseolina R. solani NS AS NS AS NS AS NS AS Control -50 18.7 50 25 75 75 18.7 12.5 Carbendazim -12.5 6.2 31.2 6.2 12.5 25 6.2 6.25 P. decumbens EPAIR6 12.5 0 0 6.2 25 18.7 0 0 P. nigricans EPSLR4 6.2 0 0 0 31.2 18.7 6.2 0 P. regulosum EPAAR5 12.5 0 25 6.2 12.5 12.5 0 6.2 P. citrinum EPSMR1 37.5 0 25 0 12.5 25 0 0 P. lilacinum EPSMS2 25 6.2 18.7 0 6.2 50 6.2 6.2 P. purpurogenum EPSML3 50 0 12.5 6.2 6.2 25 6.2 0 P. duclauxi EPASS9 25 0 6.2 6.2 31.2 18.7 6.2 0 P. lividum EPMCL12 50 6.2 50 0 0 50 0 6.2 P. purpurogenum EPEHS7 37.5 18.7 31.2 12.5 50 31 0 0 P. restrictum EPCTS8 12.5 6.2 6.2 0 12.5 43.7 6.2 0 P. thomii EPAER11 6.2 0 6.2 0 37.5 18.7 6.2 6.2 P. purpurogenum EPAER14 37.5 18.7 31.2 12.5 50 31.2 6.2 0 P. javanicum EPSLR13 6.2 0 0 0 31.2 18.7 6.2 0 P. asperum EPHAL10 12.5 12.5 25 18.7 31.2 31.2 6.2 6.2 LSD0.05 Treatment=4.451 Pathogen=2.222 Soil Type=1.573 1. Mean values in the column showing difference greater than LSD value are significantly different at p<0.05 2. Mean values in the row showing difference greater than LSD value are significantly different at p<0.05 3. Mean values in the NS and AS column showing difference greater than LSD value are significantly different at p<0.05 NS= Natural Soil; AS=Amended Soil Table 6. Effect of endophytic Penicillium species and neem cake on the growth of sunflower in green house experiment (2017). Shoot length (cm) Shoot weight (g) Root length (cm) Root weight (g) Treatments Code # NS AS NS AS NS AS NS AS Control -32.5 38.9 3.78 6.42 5.7 10.3 0.85 1.31 Carbendazim -37.8 42.9 4.52 6.07 8.4 10.2 1.24 1.28 P.decumbens EPAIR6 44.1 62.7 3.86 10.13 7 7.6 0.86 2.13 P.nigricans EPSLR4 48.3 62.0 4.89 9.53 8.6 6.5 0.96 1.41 P.regulosum EPAAR5 45.6 64.1 4.72 9.94 6.5 6.6 0.90 1.28 P.citrinum EPSMR1 38.5 64.4 3.73 14.25 7.5 7.8 0.88 2.26 P.lilacinum EPSMS2 34.5 65.5 2.06 10.19 7.0 6.4 0.72 1.61 P.purpurogenum EPSML3 35.4 60.3 2.405 9.09 6.7 5.9 0.91 1.44 P.duclauxi EPASS9 44.1 62.7 3.86 10.13 7 7.6 0.86 2.13 P.lividum EPMCL12 34.5 65.5 2.06 10.19 7.0 6.4 0.72 1.61 P.purpurogenum EPEHS7 38.5 59 2.45 8.86 8.6 11.1 0.83 1.63 P.restrictum EPCTS8 41.5 50.0 3.62 8.18 6.1 12.7 0.67 1.86 P.thomii EPAER11 48.3 62.0 4.89 9.53 8.6 6.5 0.96 1.41 P.purpurogenum EPAER14 38.5 59 2.45 8.86 8.6 11.1 0.83 1.63 P.javanicum EPSLR13 48.3 62.0 4.89 9.53 8.6 6.5 0.96 1.41 P.asperum EPHAL10 41.5 50.0 3.62 8.18 6.1 12.7 0.67 1.86 LSD0.05 10.31 8.91 2.271 5.521 3.01 2.11 0.4581 1.071 1. Mean values in the column showing difference greater than LSD value are significantly different at p<0.05 NS= Natural Soil; AS=Amended Soil Discussion Endophytic fungi have proven to be a rich source of novel secondary metabolites with interesting biological activities and a high level of chemical diversity (Schulz & Boyle, 2005). In this study, endophytic Penicillium species isolated from cultivated and wild plant species caused growth inhibition of soil borne root rotting fungi M. phaseolina, R. solani, F. solani and F. oxysporum In vitro. Penicillium are generally considered as soil inhabitant or as contaminant of food, fruits, fibers and other starchy materials, but these findings confirm previous reports about the occurrence of Penicillium as endophyte (Korejo et al., 2014; Nicoletti et al., 2014). Furthermore, culture filtrates of these Penicillium also showed strong antifungal activity in agar disc diffusion assay, suggesting that endophytic Penicillium species as a source of new bioactive metabolites that also play their role in plants against stress tolerance (Khan & Lee, 2013). Our findings are in agreement to Korejo et al., (2014). They reported that culture filtrates of endophytic Penicillium species posses significant antifungal activity. Since the discovery of BIOCONTROL POTENTIAL OF ENDOPHYTIC PENICILLIUM SPECIES penicillin a number of drugs have been developed from fungal metabolites including endophytic Penicillium species. El-Neketi et al., (2013) has been reported five new compounds from endophytic Penicillium citrinum. From endophytic Penicillium spp., 8-methoxymellein and 5hydrooxymellein have been isolated from leaves of Alibertia macrophylla (Oliveria et al., 2009). The antifungal activity against phytopathogenic fungi by these isolates suggests that endophytic fungi are producing metabolites that may be toxic or lethal to phytopathogen. In the present study, biocontrol and plant growth promoting potential of endophytic Penicillium was also evaluated in clay pots against root rotting fungi alone or in soil amended with Neem cake using sunflower as test crop. The experiment conducted in 2016 and repeated in 2017 showed significant biocontrol potential of endophytic Penicillium against root rotting fungi and improved plant growth. Endophyte protect their host plants by different means, such as the secretion of compounds toxic to pathogens, and occasionally the distraction of the pathogen’s cellular membranes, inducing cell-death in the pathogen (Ganley 2008; Prihatini et al., 2016). Several previous research studies have reported the suppression of diseases via the inoculation of plants with commonly occurring fungal endophytes (Lee et al., 2009; Poling et al., 2008). There are also reports that endo-symbionts produce plant growth regulators, which enhance the growth of the endophyte infected plants (Khan & Lee, 2013; Rashid et al., 2012). Endophytic P. funiculosum LHL06 has also been reported to significantly ameliorated the adverse effects of salinity induced by abiotic stress, and reprogrammed soybean to better growth (Khan et al., 2011). In this study efficacy of Penicillium was increased in soil amended with neem cake against root rotting fungi infecting sunflower. Enhancement of biocontrol potential of biocontrol agent in amended soil has been reported (Mansoor et al., 2007). There is also report that Arabidopsis thaliana grown in soil amended with barley grain inocula of P. simplicissimum GP17-2 or receiving root treatment with its culture filtrate exhibited resistance to Pseudomonas syringae pv., syringae (Hossain et al., 2007). Neem (Azadirachta indica) and its by-products has been broadly reported as a potential fertilizer (Gajalakshmi & Abbasi, 2004) have been reported to control plant diseases caused by fungi and parasitic nematodes (Dubey et al., 2009; Akhtar & Mahmood, 1995). Induction systemic resistance in cotton by the neem cake against soil borne pathogen has been reported by us earlier (Rahman et al., 2016). Thus endophytic Penicillium is a source of new bioactive metabolites, which could be exploited in plant protection and also in medicine. Acknowledgement Financial assistance provided by the High Education Commission, Islamabad is sincerely acknowledged. References Afzal, S., S. Tariq, V. Sultana, J. Ara and S. Ehteshamul-Haque. 2013. Managing the root diseases of okra with endo-root plant growth promoting Pseudomonas and Trichoderma viride associated with healthy okra roots. Pak. J. Bot., 45: 1455-1460. 1627 Akhtar, M. and I. Mahmood. 1995. Evaluation of a Neem based product against root-knot nematode Meloidogyne incognita. Tests of agrochemicals and cultivars, Suppl. Annal. Appl. Biol., 16: 6-7. Ali, A., M.S. Haider, I. Khokhar, U. Bashir, S. Mushtaq and I. Mukhtar. 2011. Antibacterial activity of culture extracts of Penicillium species against soil-borne bacteria. Mycopath, 9: 17-20. Barnett, H.L. and B.B. Hunter. 1998. Illustrated Genera of Imperfect Fungi. 4th ed. TheAmerican Phytopathological Society. St. Paul. Minnesota. pp. 218. Bhardwaj, A., D. Sharma, N. Jadon and P.K. Agrawal. 2015. Antimicrobial and phytochemical screening of endophytic fungi isolated from spikes of Pinus roxburghii. Arch. Clin. Microbiol., 6: 1-9. Dubey, R.C., H. Kumar and R.R. Pandey. 2009. Fungitoxic effect of neem extracts on growth and sclerotial survival of Macrophomina phaseolina In vitro. J. Am. Sci., 5: 17-24. El-Neketi, M., W. Ebrahim, W. Lin, S. Gedara, F. Badria, H. A. Saad, D. Lai and P. Proksch. 2013. Alkaloids and polyketides from Penicillium citrinum,a endophyte isolated from the Moroccan plant Ceratonia siliqua. J. Nat. Prod., 76: 1099-1104. Fill, T.P., G.K. Pereira, R.M. Santos and E. Rodrigues-Fo. 2007. Four additional meroterpenes produced by Penicillium sp., found in association with Melia azedarach. Possible biosynthetic intermediates to austin. Z. Naturforschung B, 62: 1035-1044. Gajalakshmi, S. and S.A. Abbasi. 2004. Neem leaves as a source of fertilizercum-pesticide vermicompost. Bioresource Technol., 92: 291-296. Ganley, R. 2008. Density and diversity of fungal endophytes isolated from needles of Pinus radiata. Client Report, (12925). Forest Bio-security Research Council, New Zealand. pp. 12. www.nzfoa.org.nz/images/ stories/pdfs/ content/beneficial/42803-fbrc_endophyte_ report_ final.pdf. Gherbawy, Y.A. and R.M. Gashgari. 2014. Molecular characterization of fungal endophytes from Calotropis procera plants in Taif region (Saudi Arabia) and their antifungal activities. Plant Biosystems, 148: 1085-1092. Hossain, M.M., F. Sultana, M. Kubota, H. Koyama and M. Hyakumachi. 2007. The plant growth-promoting fungus Penicillium simplicissimum GP17-2 induces resistance in Arabidopsis thaliana by activation of multiple defense signals. Plant Cell Physiol., 48: 1724-1736. Khan, A.L. and I.J. Lee. 2013. Endophytic Penicillium funiculosum LHL06 secretes gibberellin that reprograms Glycine max L. growth during copper stress. BMC Plant Biol., 13: 86. Khan, A.L., M. Hamayun, Y.H. Kim, S.M. Kang and I.J. Lee. 2011. Ameliorative symbiosis of endophyte (Penicillium funiculosum LHL06) under salt stress elevated plant growth of Glycine max L. Pl. Physiol. Biochem., 49: 852e-861. Korejo, F., R. Noreen, S.A. Ali, F. Humayun, A. Rahman, V. Sultana and S. Ehteshamul-Haque. 2017. Evaluation of antibacterial and antifungal potential of endophytic fluorescent Pseudomonas associated with Salvadora persica and Salvadora oleoides decne. Pak. J.Bot., 49: 1995-2004. Korejo, F., S.A. Ali, H.A. Shafique, V. Sultana, J. Ara and S. Ehteshamul-Haque. 2014. Antifungal and antibacterial activity of endophytic Penicillium species isolated from Salvadora species. Pak. J. Bot., 46: 2313-2318. Lee, K.., J.J. Pan and G. May. 2009. Endophytic Fusarium verticillioides reduces disease severity caused by Ustilago maydis on maize. FEMS Microbiol. Lett., 299(1): 31-37. Lucas, E.M., M.C. Castro and J.A. Takahashi. 2007. Antimicrobial properties of sclerotiorin, isochromophilone VI and pencolide, metabolites from a Brazilian cerrado isolate of Penicillium sclerotiorum Van Beyma. Brazi. J. Microbiol., 38: 785-789. 1628 FAIZAH UROOJ ET AL., Mansoor, F., V. Sultana and S. Ehteshamul-Haque. 2007. Enhancement of biocontrol potential of Pseudomonas aeruginosa and Paecilomyces lilacinus against root rot of mungbean by a medicinal plant Launaea nudicaulis L. Pak. J. Bot., 39: 2113-2119 Nair, D.N. and S. Padmavathy. 2014. Impact of endophytic microorganisms on plants, environment and humans. Sci. World J., 2014: http://dx.doi.org/10.1155/2014/250693. Nash, S.M. and W.C. Snyder. 1962. Quantitative estimations by plate counts of propagules of the bean root rot Fusarium in field soils. Phytopath., 52: 567-572. Nicoletti, R., A. Fiorentino and M. Scognamiglio. 2014. Endophytism of Penicillium species in woody plants. The Open Mycology J., 8: 1-26 Nicoletti, R., M.P. Lopez-Gresa. E. Manzo, A. Carella and M.L. Ciavatta. 2007. Production and fungitoxic activity of Sch 642305, a secondary metabolite of Penicillium canescens. Mycopathologia, 163: 295-301. Oliveira, C.M., G.H. Silva, L.O. Regasini, L.M. Zanardi, A.H. Evangelista, M.C.M. Young, V.S. Bolzani and A.R. Araujo. 2009. Bioactive metabolites produced by Penicillium sp. 1 and sp. 2, two endophytes associated with Alibertia macrophylla (Rubiaceae). Z. Naturforsch. C, 64: 824-830. Poling, S.M., D.T. Wicklow, K.D. Rogers and J.B. Gloer. 2008. Acremonium zeae a protective endophyte of maize, produces dihydroresorcylide and 7–hydroxydihydroresorcylides. J. Agric. Food Chem., 56: 3006-3009. Prihatini, I., M.Glen, T.J. Wardlaw and C.L Mohammed. 2016. Diversity and identification of fungi associated with needles of Pinus radiate in Tasmania. Southern Forests: J. Forest Sci., 78: 19-34. Qureshi, S., K. Ruqqia, V. Sultana, J. Ara and S. EhteshamulHaque. 2012. Nematicidal potential of culture filtrates of soil fungi associated with Rhizosphere and rhizoplane of cultivated and wild plants. Pak. J. Bot., 44: 1041-1046. Qureshi, S.A. 2003. Studies on Antibiotics from Soil Fungi. Ph.D. thesis. Department of Biochemistry, University of Karachi, Karachi, Pakistan. Rab, J.A., M.Z. Iqbal, M. Shafiq and M. Athar. 2016. Studies on vegetation and soil characteristics of Karachi University campus. Scholar J. Res. Agric. Biol., 1: 20-28. Rahman, A., V. Sultana, J. Ara and S. Ehteshamul-Haque. 2016. Induction of systemic resistance in cotton by the neem cake and Pseudomonas aeruginosa under salinity stress and Macrophomina phaseolina infection. Pak. J. Bot., 48: 1681-1689. Raper, K.B. and C. Thom. 1949. A Manual of the Penicillia. The Williams & Wilkins Company, Baltimore, pp. 875. Rashid, S., T.C. Charles and B.R. Glick. 2012. Isolation and characterization of new plant growth-promoting bacterial endophytes. Appl. Soil Ecol., 61: 217-224. Schulz, B. and C. Boyle. 2005. The endophytic continuum. Mycol. Res., 109: 661-686. Shafique, H.A., R. Noreen, V. Sultana, J. Ara and S. Ehteshamul-Haque. 2015. Effect of endophytic Pseudomonas aeruginosa and Trichoderma harzianum on soil-borne diseases, mycorrhizae and induction of systemic resistance in okra grown in soil amended with Vernonia anthelmintica L. seed’s powder. Pak. J. Bot., 47: 2421-2426. Sheikh, A.H. and A. Ghaffar. 1975. Population study of the sclerotia of Macrophomina phaseolina in cotton fields. Pak. J. Bot., 7: 13-17. Singh, S.B. 2003. Isolation, structure and HIV-1 integrase inhibitory activity of anthoviridicatin E and F, two novel fungal metabolites produced by Penicillium chrysogenum. Helv. Chem. Acta, 86: 3380-3385. Stierle, A.A., D.B. Stierle and K. Kelly. 2006. Berkelic acid, a novel spiroketal with selective anticancer activity from an acid mine waste fungal extremophile. J. Organic Chem., 71: 5357-5360. Visagie, C.M., J. Houbraken, J.C. Frisvad, S.B. Hong, C.H.W. Klaassen, G. Perrone, K.A. Seifert, J. Varga, T. Yaguchi and R.A. Samson. 2014. Identification and nomenclature of the genus Penicillium. Stud. Mycol., 78: 343-371. Wilhelm, S. 1955. Verticillium wilt of the strawberry with special reference to resistance. Phytopath., 45: 387-391. (Received for publication 14 July 2017)