Optimization of culture conditions for Paecilomyces lilacinus ( Thom ) Samson M-14

Paecilomyces lilacinus is known as an effective parasite on nematodes which cause diseases to plants. P. lilacinus shows potential as a biocontrol agent against plant parasitic nematodes. The objective of this study is to optimize culture methods including nutritional requirements and environmental factors. The optimized culture conditions for biomass yields of P. lilacinus M-14 were spore suspension on basal medium (sucrose 19.00 g, soy peptone 4.06 g, K2HPO4 1.00 g, KCl 0.50 g, MgSO4 0.50 g, FeSO4 0.01 g and 17.00 g Bactor) for the first stage culture of 4 days under room condition for fungal growth, and then moved to another medium (maltose 5.00 g, soy peptone 2.50 g, ZnSO4·7H2O 0.25 gL -1 , Na2MoO4·2H2O 0.005 gL -1 , H3BO4 0.005 gL -1 , CuSO4·5H2O 0.01 gL -1 and 17.00 g Bactor) for another 4 days culture. The environmental factors combination was water potential -1.2 MPa/pH 3/light 12 h/temperature 29°C for biomass yields, and for sporulation of P. lilacinus M-14 under the environmental conditions, it was water potential -1.2 MPa/pH 3/24 h light/29°C. It will provide valuable insight into culturing of the biocontrol fungus.


INTRODUCTION
With the increase in awareness of the harmful effects of chemical pesticides and the changing public attitude towards environmental pollution, chemical pesticides is losing their popularity among farmers (Pandey et al., 2000;Anastasiadis et al., 2008).Environmental concerns for the quality of the environment and food safety have created social and legislative pressure to remove many agricultural pesticides from the market (Noling and Dickson, 1992;McKenry et al., 1994).Biological control is considered as the most safe and effective alternative tochemical control methods (Kutschera and Hossfeld, 2012;Sharma et al., 2014;Liu et al., 2017).Biocontrol agent, like Paecilomyces lilacinus, is a soil-inhabiting fungus that has shown great potential (Morgan-Jones et al., 1984;Jatala, 1986;Dube and Smart, 1987;Khan et al., 2006, Kepler et al., 2017;Chaverri et al., 2015;Yu et al., 2013), which has been reported to reduce Meloidogyne incognita (one of the most destructive pests of a wide range of crops, causing more than 10% loss in the world's total crop production) populations and this the *Corresponding author.E-mail: lgao@ippcaas.cn.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License world's total crop production) populations and this reduction was showed in the tomato yield (Lara et al.,1996;Topp et al., 1998).Mani et al. (1989) found that it has good biocontrol efficiency on many root-knot nematodes.
The aim of this study is to optimize the biomass yields of P. lilacinus based on its growth on a broad culture medium, such as PDA, PDB, PCA, Czapek and which have better growth and sporulation on Czapek, after 8 days culture, and has 1.10 g 50 mL -1 mycelia and 28.50×10 6 mL -1 spore yields (Li et al., 2005).Suebsak (1996) found that the optimal culture medium was the juice of soy or potato, which contain more than 0.4 M Mg 2 ＋ and 0.1×10 -3 M Cu 2＋ and have great suppression on the growth of this fungi.P. lilacinus could also grow on nature basis, including plant leaf, rice, wheat, and green pea (Mani et al., 1989;Abu-Laban and Saleh, 1992).Different nutrition leads to different mycelia and spore yields; it could produce more spores on rice than a green pea (Zaki and Bhatii, 1991;Xue et al., 2013).Siddiqui and Mahmood (1994) reported that the leaf extracts and residues of Peristrophe bicalyculata and Dalbergia sissoo were best as culture substrates in 17 plants for P. lilacinus in the fields.Villanueva and Davide (1984) found that better mycelia growth was acid as compared to alkalescence, with a normal growth at 15-35°C, and a better growth and sporulation at 25-30°C.Suebsak found that the optimal culture medium was the juice of soy or potato under the temperature of 31°C with 220 to 270 r min -1 (Sun et al., 1997).Relative humidity is the key to germination of P. lilacinus, when RH reaches 85%, it began to germinate, with the highest germination at 98% RH under 25°C (Huang et al., 1994).
The combination effects of culture conditions, including nutrition and environmental factors on the growth and sporulation of P. lilacinus is reported in this study.This method is different from previous reports (Gao et al., 2009).This information will provide more details on the fungus' mass production.

Fungal strain
The tested nematophagous fungus, P. lilacinus M-14 was originally isolated from Heterodera glycines from Heilongjiang (China), and deposited in the CGMCC in Institute of Microbiology, CAS.
The basal medium included 17.00 g Bactor (Difco) agar, sucrose 19.00 g (equal to 8 g carbon), soy peptone 4.06 g (equal to 0.33 g nitrogen), K2HPO4 1.00 g, KCl 0.05 g, MgSO4 0.50 g, FeSO4 0.01 g per liter.This medium was used for the first stage culture for 4 days.

Effects of carbon concentrations and carbon to nitrogen ratios
Sucrose (42% carbon): 1, 2, 4, 8 and 16 gL -1 was used to adjust carbon concentrations, soy peptone (8% nitrogen): 0.2, 0.4, 0.8 and 1.6 gL -1 was used to adjust nitrogen concentrations, which resulted in C:N ratios ranging from 0.625:1 to 80:1.This was used for the second stage culture for sporulation for another 4 days.The optimal carbon concentration of 2 gL -1 with carbon to nitrogen ratio of 10:1 was obtained (Gao and Liu, 2009).

Effects of carbon and nitrogen sources combination
The combinations of sources include maltose, sucrose, starch soluble, soy peptone and yeast extract.Based on the carbon concentration, 2 gL -1 and C/N ratio of 10:1, the combinations of different carbon and nitrogen sources for sporulation with this novel method was obtained.For each combination, they were added to the basal medium to replace the sucrose and soy peptone as sporulation medium for the second stage culture of more 4 days.The basal medium for sporulation of another 4 days was used as a control.

Effects of mineral elements
After testing the components and concentration gradients of six mineral elements for sporulation of these two isolate with onefactor-at-a-time method, the optimal components for the sporulation of P. lilacinus M-14, including ZnSO4•7H2O 0.25 gL -1 , Na2MoO4• 2H2O 0.005 gL -1 , H3BO4 0.005 gL -1 , and CuSO4• 5H2O 0.01 gL -1 was obtained.

Effects of environmental conditions on sporulation of P. lilacinus M-14 using the method
The two-stage cultivation method in the plates was used to evaluate the effects of pH, water potential, dark/light cycle and temperature on the second stage culture of 4 days more on sporulation of the biocontrol fungi.Water potential includes -0.3, -0.8, -1.2, -2.1,-3.9 and -7.3 MPa; pH includes 3, 4, 5, 6, 7, 8, 9, dark/light cycle includes 24/0 h, 12/12 h, 0/24 h, temperature includes 20, 23, 26, 29 and 32°C.In this study, two better levels of the orthogonal experiment were selected as shown in Table 1.

Optimization of the culture conditions
After the nutrition combination by full experiment, the combination of nutrition together with environmental factors was optimized for sporulation of P. lilacinus M-14 by L16(2 15 ).

Statistical analysis
One-way analysis of variance (ANOVA) was used.Duncan's multiple range test was done using Statistical Analysis System (Version 8.2, SAS Institute, Cary, NC) to test the significant differences at P = 0.05.

The sources combination of carbon and nitrogen
The combination of carbon and nitrogen sources on  2).The combination of maltose and soy peptone showed the best sporulation.

Optimization of the conditions
According to the four factors and two levels shown in Table 1, L 16 (2 15) was used to optimize the experimental conditions (Table 3).Based on the orthogonal method, the results are showed in Table 3, and the order of effects of all factors on mycelia growth could be determined as 32.12 (water potential) > 16.62 (pH) > 6.71 (light) > 4.29 (temperature) according to R (maximum difference) in Table 4.

Cultivation with two-stage method
The method was used to optimize the biomass and sporulation of nematophagous fungus separately.With the help of membranes of cellophane, the basal medium for first stage of 4 days for fungal growth was transferred to the second stage of 4 days sporulation culture.The nutrition and two better levels of 4 environmental factors on sporulation of P. lilacinus M-14 were then combined by orthogonal matrix method to obtain better combinations.

Effects of carbon and nitrogen sources
Some nutritional components could accelerate the sporulation of P. lilacinus M-14, while their combination may not be the best for its sporulation.These results proved this phenomenon, which also indicated that the full experiment of nutrition was necessary, and also the orthogonal method was essential for the sporulation of P. lilacinus M-14 on nutrition and environmental factors.

Optimization by orthogonal matrix method
Based on the results, the research combined the nutritional components environmental factors, which is different from other reports which only referred to one fields.In this study, water potential played an important role in biomass, while pH was the key to sporulation.

Combinations of the three fields
The nutritional components can significantly influence growth and sporulation of many fungi (Culbreath et al., 1986;Tiganomilani et al., 1995;Rao et al., 1997), which showed the essential to optimize nutrition for the fungus.The nutrition for the fungal biomass and sporulation may not necessarily correlate under the orthogonal matrix method, which also indicated the essential of two-stage method.

Basis for formulation and storage conditions
Instability of the biofungicide greatly limited their *Biomass yields (mg per colony).† Sporulation (105 conidia per colony).K1 and K2 are the total content of biomass yields from the level 1 and level 2 separately; k1 and k2 are the mean value of levels 1 and 2 separately.K1' and K2' are the total spore yields from the level 1 and level 2 separately; k1' and k2' are the mean value of levels 1 and 2 separately.R is the maximum of k1, k2 minus the minimum of k1, k2 and R' is the maximum of k1, k2 minus the minimum of k1, k2 respectively.O is the optimal level of biomass yields and O' is the optimal value of spore yields.application, with better nutrition and environmental storage conditions helping in their resistant to unfavorable conditions (Miller et al., 1997).These results could also help in selecting better material to formulate for a longer shelf life (for example, some fungi are sensitive to light, and the material resistant to light can be chosen).In addition, chitin helps in stabilizing the fungi and also for a better activity in microbiology with a better control efficiency in pests (Rodriguez et al., 1984;Sun et al., 1997).

Conclusion
In summary, the culture conditions for biomass yields and sporulation of P. lilacinus M-14, were optimized, which will provide valuable information on mass production (both yields of biomass and spore) of the potential biocontrol agent.

Table 1 .
Effects of environmental factors on sporulation of P. lilacinus M-14.

Table 2 .
Effect of carbon and nitrogen source on the sporulation of P. lilacinus M-14 (10 5 /ml).
c sporulation of the isolates showed significant effects (Table

Table 4 .
Analysis of environmental factors on biomass production and sporulation of P. lilacinus M-14 with this novel method.

Table 5 .
The variance analysis of L16(2 15 ) orthogonal test on optimation of environmental factors for biomass yields and sporulation of P. lilacinus M-14.

Table 6 .
Effects of combinations of environmental factors on biomass yields and sporulation of P. lilacinus M-14.