FUNGAL CONTAMINANTS THREAT OYSTER MUSHROOM (Pleurotus Ostreatus (Jacq

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FUNGAL CONTAMINANTS THREAT OYSTER MUSHROOM (Pleurotus ostreatus (Jacq. Ex Fr) Kummer) CULTIVATION I Made Sudarma*, Ni Made Puspawati dan Gede Wijana*

*Department of Agroetechnology, Faculty of Agriculture, Udayana University, Jl. PB. Sudirman Denpasar-Bali. E-mail: [email protected]. HP. 08123639103

ABSTRACT One of the causes of failure of the cultivation of oyster mushroom (Pleurotus ostreatus (Jacq. Ex Fr) Kummer) is still much contamination baglog inhibit growth and cause failure of oyster mushroom production. For that study was conducted to determine fungal contaminants in the baglog media and inhibiting ability against oyster mushrooms in vitro. Research carried out by the observation methods, sampling randomly contaminated baglog 10-20% of the amount of contaminated baglog, repeated 3 times. Study to be implemented in venture oyster mushroom address: Jl. Siulan gang Zella no. 7 Denpasar, from April to August 2014. The results showed that air-borne fungus could potentially cause failure of oyster mushroom cultivation. The highest prevalence was found in Fusarium spp. (25.6%), while the highest inhibition was found in Mucor spp.(94.7±8.5). Fungal contaminants originating from baglog, the most dominant with the highest prevalence was Trichoderma spp (35.71%). This fungus was very dangerous for the survival of oyster mushroom cultivation.

Keywords: Oyster mushroom (Pleurotus ostreatus (Jacq. ex Fr) Kummer), inhibiting ability, and the prevalence of fungal contaminants.

INTRODUCTION

Development of oyster mushroom cultivation particularly in Bali received threats by a number of fungal contaminants. Fungal contaminants can originate from the air and sawdust media. Green mold caused by Trichoderma spp. is a major disease that is found in oyster mushroom (Kredic et al., 2010). Fungus isolated and characterized from compost include such as Aspergillus, Trichoderma, Mucor, Penicillium, Alternaria, Cladosporium, Monilia, Helminthosporium, Coccidioides, and Scedosporium (Ashraf et al., 2007). In Chiapas, Mexico, the most common contaminants during spwan phase were: Streptomyces sp., Penicillium sp. Aspergillus ochraceus, A. flavus, Cunninghamella sp., and Trichoderma viride. During the incubation phase Monilia sp. and T. viride were found. During fructification phase the most abundant contaminants were: Poronia sp. and Coprinus sp. (Lopez-Arevalo et al., 1996). In Indonesia, fungal contaminants were found

1 to inhibited the growth of oyster mushroom was Neurospora spp., Trichoderma spp., Mucor spp., and Penicillium spp. (Anggrianto, 2012). The quality of the growing media (compost), the number of spores in the air, and the phase of growth of oyster mushrooms determine the severity of contamination on baglog (Anastasi et al., 2005). The density of spores in the air , accompanied by a lot of opportunities for contamination causing the failure of oyster mushroom cultivation. Fungi contaminants that have been known to cause fungal growth failure of oyster mushroom was: Neorospora spp., Trichoderma spp., Mucor spp., and Penicillium spp (Anggrianto, 2012). Fungal contaminants on the media type and inhibiting ability to the growth of oyster mushroom in vitro until now unknown, it is necessary for in-depth studies to answer the problems mentioned above.

MATERIALS AND METHOD

The place and time study The study was conducted in Breeding and Development Company Oyster Mushrooms with address Siulan street, Zella alley No. 7 Denpasar. The application time at April 2014 until November , 2014.

Making media baglog The composition of the media used were wood dust : bran : corn : limestone (CaCO3 ) : NPK at a ratio of 100 : 10 : 5 : 2.5 : 1. The process of making was, (1) All the ingredients are mixed while adding water. The amount of water that was adapted to medium compact when clenched does not decompose and when it does not remove the water squeezed . (2) A total of 0.5 kg of medium then fed into a heat resistant plastic the size of 1 kg and then pressing and tie it closed with the use of rubber slipping cotton on top. (3) Sterilization 5 hours . After sterile, store in a clean room . (4) After a cold was inoculated with seedling (F3).

F3 seedlings F3 seedling of oyster mushroom were taken from the Breeding and Development Company Oyster Mushrooms with address Siulan street and Zella alley No. 7 Denpasar. F3 seedlings using material from corn that is inserted in a bottle of beer, and it was inoculated with F2 seeds, once the seeds are fully grown mycelium , then used as F3 seedling.

Inhibition test of fungi contaminants against Oyster Mushrooms Each fungus contaminants tested for inhibitory against the growth of oyster mushroom with a dual culture technique. Percent inhibition can be calculated using the following formula (Dolar, 2001; Jayalal and Adikaram, 2007; Mojica-Marin et al., 2008):

A – B % inhibition = x 100 A

A = Oyster mushroom colony diameter in the control (mm) B = Oyster mushroom colony diameter in treated (mm).

Study of air-borne fungi Study of air -borne fungus is done by placing 5 Petri dishes containing PDA (mixture of potatoes 200 g , 15 g sugar, 20 g in order to 1000 ml of distilled water) and livoploxasin (antibacterial antibiotics) with a concentration of 0.25 % (w/v). Five Petri dishes were placed openly at work ( where do the activity), and placed from 7:00 am to 13:00 noon ( note the average work activity during these hours ). Thereafter , the Petri dish was closed and incubated in a cabinet incubator for 2 days , until the fungus appear in Petri dishes , and counted the number of colonies. Furthermore , each colony was purified again by moving to a new Petri dishes containing PDA medium. Incubation of media in the dark at room temperature (27±2oC). Isolates were identified macroscopically after the age of 3 days to determine colony color and growth rates , and the identification of microscopically to determine the septa in hypha, forms spores / conidia and sporangiophore. Identification of fungi using reference books such as Samson et al, 1981; Pitt and Hocking, 1997; Barnett and Hunter, 1998; and Indrawati et al., 1999). Study of air-borne fungus done 3 times. Thereafter, air -borne fungi tested for inhibiting ability against oyster mushrooms with the formula as mentioned above.

Study of fungal contaminants derived from baglog A total of 20% baglog as samples taken from 50 baglog contaminated, and repeated 3 times. Baglog contaminated characterized by a change in color at the top of baglog, This section was taken (0.1 g), then placed in Petri dishes containing PDA (mixture of potatoes 200 g, 15 g sugar, 20 g in order to 1000 ml of distilled water) and livoploxasin (antibacterial antibiotics) with a concentration of 0.25% (w/v). Furthermore, isolates identified macroscopically after 3 days old. Fungi contaminants derived from baglog tested for inhibiting ability against oyster mushroom with the formula as mentioned above.

RESULTS AND DISCUSSION

Prevalence of air-borne fungus and fungal contaminants derived from baglog The results of the capture of spores using Petri dishes with 5 replications obtained 24±2 cfu/a Petri dish. Air-borne fungi identification such as 10 isolates of Aspergillus sp., 7 isolates of Aspergillus niger, one isolate of Brachysporium sp., one isolate of Cunninghamella sp., 19 isolates of Fusarium sp., one isolate of Geotrichum sp., 8 isolates of Neurospora sp., 18 isolates of Mucor spp., 2 isolates of Penicillium sp., one isolate of Umbelopsis sp., 5 isolates of Stachybotrys sp., and one isolate of Trichoderma sp. (Table 1, Figure 1). Fusarium sp. dominated by the prevalence (frequency isolates) i.e. 25.7 % , followed by Mucor spp. 24.3 %, Aspergillus spp. 13.5 %, Neurospora spp., 10.8 %, A. niger, 9.5%, Stachybotrys spp. by 6.7 % , while the other 1.4 % respectively (Table 1).

Table 1. Number of air -borne fungus, fungal contaminants derived from baglog and prevalence

No. Fungus Air-borne fungus Fungal contaminants derived from baglog Number Prevalence Number of Prevalence of (%) isolates (%) isolates 1. Aspergillus spp. 10 13.5 9 21.43 2. A. niger 7 9.4 - - 3. Brachysporium sp. 1 1.4 - - 4. Cunninghamella sp. 1 1.4 - - 5. Fusarium spp. 19 25.6 1 2.38 6. Geotrichum sp. 1 1.4 - - 7. Gliocladium sp. - - 1 2.38 8. Mucor spp 18 24.3 2 4.76 9. Neurospora spp. 8 10.8 4 9.52 10. Paecilomyces sp. - - 1 2.38 11. Penicillium spp. 2 2.7 6 14.28 12. Pythium sp. - - 1 2.38 13. Stachybotrys spp. 5 6.7 2 4.76 14. Trichoderma spp. 1 1.4 15 35.71 15. Umbelopsis sp. 1 1.4 - - 74 42

Trichoderma sp. Stachybotrys spp. Umbelopsis sp. Penicillium spp. Mucor spp. Neurospora spp. GeotrichumOidium spsp.. Fusarium spp. Cunninghamella sp. Brachysporium sp. A. niger Aspergillus spp.

0 5 10 15 20 25 30

Figure 1. Prevalence of air-borne fungus that could potentially be a fungal contaminant

Based on Table 1, 7 of the 10 species of fungi contaminants derived from the baglog same with air-borne fungus , this means that 70% fungal contaminants derived from air-borne fungus, and the rest (30%) comes from the substrate baglog. Fungal contaminants from contaminated baglog media, including Trichoderma spp. dominate with a prevalence of 35.71%, followed by Aspergillus spp . amounted to 21.43% , Penicillium spp . amounted to 14.28% , Neurospora spp. amounted to 9.52%, Mucor spp . amounted to 4.76% , while the Fusarium sp., Gliocladium sp., and Paecilomyces sp., each with a prevalence of 2.38% (Table 1; Figure 2). Trichoderma spp., was the most virulent fungus with the ability to survive and thrive in compost media (materials baglog), which has a mechanism of inhibition , competition space and nutrients , mycoparasitic , and antibiosis. Cellulolytic fungi such as Aspergillus , Penicillium and Trichoderma associated with the composting process and can speed up the composting for re- cycle efficiency. This fungus can cause disease in oyster mushroom known as green mold disease (Sharma et al., 2007). Omokaro and Ogechi (2013) states that the fungus was isolated from oyster mushroom growing substrate media include Aspergillus (prevalence 40.9%), Fusarium (22.7%), Mucor (5.6% ), Penicillium (17,0%), Rhizopus (11.6%) and Trichoderma (2.3%). Likewise, the results Wickremasinghe et al . (1999) in Sri Lanka, has been isolated and identified the fungi from compost substrate such as Aspergillus fumigatus, Chetomium thermophile , Mucor pusillus and Trichoderma harzianum.

Trichoderma spp. Stachybotrys spp. Pythium sp. Penicillium spp. Paecilomyces sp. Neurospora spp. Mucor spp. Gliocladium sp. Fusarium sp. Aspergillus spp.

0 5 10 15 20 25 30 35 40

Figure 2. Prevalence of fungal contaminants derived from baglog

Trichoderma spp . attack the host through the growth of hypha throughout the host and through the establishment of structures resembling papilla, growing freely on its host. Structure that resembles the papilla of Trichoderma spp., can also form glycerol, as the transcription of genes involved in lipid catabolism and increase osmoregulation during the contact stage mycoparasitism (Druzhinina et al., 2011) . Symptoms of contaminated baglog by Trichoderma sp . was green gradually creeping into the black. Symptoms begin to appear at the top of baglog and evolve towards the bottom. This means that the contamination caused by air–borne fungi when seed planting into baglog. Baglog that contaminated different colored with healthy baglog (white). Trichoderma hypha show branched, with phialide growth of conidiophores and conidia support (Figure 3).

Inhibition of air-borne fungus and fungal contaminants derived from baglog Percent inhibition of air-borne fungus showed that Mucor spp. highest i.e. 94.7±8.5%, followed by Aspergillus niger (93.95±9.7%), Stachybotrys spp. (92.59±1.51%), Neurospora spp. (91.67±914%), Trichoderma sp., and Geotrichum sp. respectively 88.89±0.1%, Aspergillus spp . (83.88±478%), Penicillium spp., and Umbelopsis sp. respectively by 77.78±0.1%, whereas others such as Brachysporium sp., and Cunninghamella sp. showed no inhibitory effect on the growth of oyster mushroom mycelium in vitro (Table 2).

A B C

sp f knf

D 20 µm

Figure 3. (A) Symptom of baglog was contaminated by Trichoderma sp., (B) baglog health (control), (C) mycelium of Trichoderma sp. in Petri dish, and (D) observation microscopically with magnitude 400x (sp = spore, f = phialide, and knf = conidiophore)

Inhibition of fungal contaminants derived from baglog such as Aspergillius spp. i.e. 80.97±8.17%, Fusarium sp. (72.22±0.2%), Gliocladium sp. (70±0.1%), Mucor spp. (91.11±12.57), Neurospora spp. (82.22±1.7%), Paecilospora sp. (82.22±0.2%), Penicillium spp. (84.33±5:02), Pythium sp. (83.33±0.1%), Stachybotrys spp. (78.71±9.71%), and Trichoderma spp. (84.96±7.36%) (Table 2). The mechanism of inhibition by air -borne fungus was found almost entirely in the competition (competition space and nutrients), except Trichoderma sp. was found antibiosis and competition. While the mechanism of inhibition of fungal contaminants originating from baglog almost of all in antibiosis mechanism, except Stachybotrys spp . show competition space and nutrients.

Table 2. Percent inhibition of air-borne fungus and fungal contaminants derived from baglog

No. Fungus Percent inhibition Air-borne fungus Fungal contaminants derived from baglog 1. Aspergillus spp. 83.88±4.78 80.97±8.17 2. Aspergillus niger 93.95±9.72 - 3. Brachysporium sp. - - 4. Cunninghamella sp. - - 5. Fusarium spp. 85.14±9.6 72.22±0.2 6. Geotrichum sp. 88.89±0.1 - 7. Gliocladium sp. - 70±0.1 8. Neurospora sp. 91.67±9.14 82.22±1.7 9. Mucor spp. 94.7±8.5 91.11±12.57 10. Paecilospora sp - 82.22±0.2 11. Penicillium spp. 77.78±0.1 84.33±5.02 12. Pythium sp. - 83.33±0.1 13 Umbelopsis sp. 77.78±0.1 - 14. Stachybotrys spp. 92.59±1.51 78.71±9.71 15. Trichoderma sp. 88.89±0.2 84.96±7.36

Ashraf et al . ( 2007) found that the fungus associated with oyster mushroom growing medium (compost) such as Aspergillus, Trichoderma, Mucor, Penicillium, Alternaria, Cladosporium, Monilia, Helminthosporium , Coccidioides, and Scedosporium. This means that the air -borne spores mostly have the potential harm oyster mushrooms media. Sharma et al . (2007 ) the results of his research in India showed that the fungus Aspergillus spp., Aspergillus niger, Fusarium spp., Mucor spp. and Trichoderma spp. were competitor fungus or may cause disease in cultivated mushrooms such as oyster mushrooms. Lopez - Arevalo et al . ( 1996) also found that Penicillium sp., Aspergillus sp., Trichoderma sp. and Cunninghamella sp. a fungal contaminant in a tropical country of Mexico.

CONCLUSION

Air-borne fungus could potentially cause failure of oyster mushroom cultivation. The highest prevalence was found in Fusarium spp., while the highest inhibition was found in Mucor spp. Fungal contaminants originating from baglog, the most dominant with the highest prevalence was Trichoderma spp . This fungus was very dangerous for the survival of oyster mushroom cultivation.

Acknowledgements Authors wish to thank to the Rector of Udayana University for their assistance and the opportunity given so that research can be resolved, Dean of the Faculty of Agriculture, Udayana University, and Chairman of the Institute for Research and Community Service Udayana University, for their help and cooperation so that research can be funded to completion.

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