Evaluation of Substrates for Mass Culture of Phaeoacremonium parasiticum, the Fungus Responsible for Artificial Oleoresin Deposition in Agarwood Plant

For establishing the favourable media for growth of Phaeoacremonium parasiticum, the fungus was inoculated in five different media (Potato Dextrose Broth (PDB), Host Extract (50%) + PDB (50%) (HE + PDB), and the solid media viz., Rice Bran (RB), Maize Meal (MM) and Wheat Bran (WB)) at pH of 6.5 and incubated at a temperature of 30 ̊C ± 1 ̊C. The solid media were added with water at the rate of 70 ml/100g of solid substrate. After 30 days of inoculation, conidial population of P. parasiticum in liquid media was lower compared to conidial population in solid media. In solid media, MM media showed the highest conidial population (9.56 log∙cfu/g) followed by WB (9.50 log∙cfu/g) and RB (9.38 log∙cfu/g). Hence, Maize Meal media (MM: Water = 100 g:70 ml) at pH of 6.5, incubation temperature: 30 ̊C ± 1 ̊C for 30 days would be the standard technique for mass production of P. parasiticum.

More modern studies confirmed that, Agarwood has bioactive products that function as effective anti-microbial compounds. Agarwood may have anticancer activity, and can be used as an antidepressant and used to promote good health [4] [5]. The global perfume industry has become interested product, and Agarwood has also caught the interest of esoteric circles of non-Asian societies. Agarwood has become the most expensive perfumery raw material in the world and its value surpasses the value of gold [6]. The price of Agarwood is from few dollars per kilogram to over 30,000 US dollars for top quality resinous wood in international market [7]. Seeking and cutting down the tree for harvesting Agarwood has made the Agar trees in nature reduced. To meet the demand of supplying to the market, lots of Agar plants have been grown from small scale at farmer house to large scale in farm.
Even they have highly useful and highly economic value, but unfortunately, in natural forests, the Agar tree only produces oleoresin when it is infected by fungus and only 7% -10% of the trees are infected by the fungus [8]. The other report showed that in fact, only 1% of Agar trees in nature contain the resinous Agarwood substance [9]. Currently, various researches and investigations concluded that the oleoresin forming in Agar tree relates to the certain contaminated factors combine with certain conditions [2]. The resin production is response to fungal infection and Phaeoacremonium parasiticum is one of the known fungi reported to be associated with Agarwood formation [1] [10] [11].
Its pathogenesis was confirmed and the technique for its isolation and in-vitro culture method has been developed. The result also showed that conidial population density of P. parasiticum in Potato Dextrose Broth was 6.24 log•spore/ml and material used for mass culture was costly [12]. Hence, study of evaluation of substrates for mass culture of Phaeoacremonium parasiticum was done to achieve the maximum potential sporulation of P. parasiticum with acceptable cost.

Calculation of Colony Forming Unit (CFU)
The colony forming units per petriplate were counted with the help of the marker (25 petriplates were counted per treatment). Number of colony forming unit per g or ml of sample was calculated follow the formulation [13] as below and converted log values.
Number of colony forming unit/g or ml of sample = (Number of cfu/plate) × (reciprocal of dilution of sample).

Results and Discussion
Phaeoacremonium Ajello et al. [14] showed cardinal temperatures for P. parasiticum growth as 15˚C (minimum), 25˚C (optimum), and 35˚C (maximum). The other study suggested that discrepant optimal growth temperatures have been obtained for P. parasiticum, ranging from 25˚C -30˚C and the optimum was closer to 30˚ than to 25˚C [15]. Akhila et al. [12], after trying incubating P. parasiticum at temperature viz., 25˚C ± 1˚C, 27˚C ± 1˚C, 30˚C ± 1˚C and 32˚C ± 1˚C in PDA media, concluded that optimal growth temperature of P. parasiticum was 30˚C ± 1˚C. Also in the same study, the optimum growth pH of P. parasiticum was suggested at 6.5 after trying at pH viz., 5.0, 5.5, 6.0, 6.5 and 7.0. Based on the previous studies, the temperature for growth of P. parasiticum was set at 30˚C ± 1˚C and pH was set at 6.5 for the present study.
The result of population density (cfu/g or ml) count showed that, in liquid media, PDB supported the highest growth with 7.23 log•cfu/ml followed by HE + PDB with 4.85 log•cfu/ml. MM media as solid media supported for maximum conidial population (9.56 log•cfu/g) of the fungus, followed by WB (9.50 log•cfu/g) and RB (9.38 log•cfu/g) ( Table 1 and Figures 2(a)-(d)). The result of liquid media (PDB and HE + PDB) observed in the present study is in accordance with the study by Akhila et al. [12]. They also tried mass culture of P. parasiticum in liquid media, and after 20 days of inoculation, the spore population in PDB was higher than the spore population in HE + PDB. Zhang et al. [16] reported that the Host Extract (HE) from the healthy part of Agar tree contains a low amount of Agawood oil compound as a defence agent against P. parasiticum. Hence, in the HE media or in the added HE media, the P. parasiticum growth was lower compared to the growth in other media. This result is in agreement with various studies which stated that, the Agarwood extract originating from Agar trees exhibited strong antifungal activity against Fusariumsolani, F. oxysporum, Candida albicans, Lasiodiplodia theobromae [16] [17] [18] [19]. Mass culture of Beauveria bassiana was done by the several workers. According to each study, the best result of spore production in different media was T. K. L. Le et al.  showed as: in Rice flour liquid media (2.4 × 10 8 spores/ml), and in Rice media (6.24 × 10 4 spores/g) [20], in Cowpea solid media (9.06 × 10 7 spores/g) [21].
In mass culture production of Deuteromycetes fungi, solid media viz., Rice Bran (RB), Maize Meal (MM), Wheat Bran (WB) always are considered to be used for study because they are the common products, available, relatively cheaper and supporting both mycelia growth and spore production. Among those, Rice or Rice Bran usually gives the better result [24] [25] [26]. But in the present study, for mass culture of P. parasiticum, MM showed as the best substrate followed by WB. Maize contains high proportions of carbohydrates (starch), and to a lesser extent minerals, fats and proteins [27]. Lancey [28] stated that, because the whole maize is a good source of thiamin, pyridoxine and phosphorus, and a fair source of riboflavin, niacin, folate, biotin, iron and zinc, so maize meal is one of the favourite substrate for fungi development that many cause nutritional losses and production of toxic substances known as mycotoxin. According to Pollack and Benham [29], Maize meal was a well-established mycological medium used for the cultivation of fungi and to study chlamydospores production of various fungi.
Wheat Bran, a by-product of the dry milling of common Wheat, is one of the major agro-industrial by-products used in animal feeding. It consists of the outer layers (cuticle, pericarp and seedcoat) with small amount of starchy endosperm of the Wheat kernel. Wheat Bran has been used in fungal culture in laboratory as well as fungal mass culture in industrial scale [30].
The present study showed that, Maize Meal media (MM: Water = 100 g:70 ml) at pH of 6.5, incubation temperature: 30˚C ± 1˚C for 30 days would be the standard technique for mass production of P. parasiticum.