Effect of different pH values on growth and sporulation of Estye vermicola

Esteya vermicola, an endoparasitic fungus of pinewood nematode, exhibits great potential as a biological agent against pinewood nematodes. In order to improve the growth rate, sporulation and proportion of lunate conidia, the optimization of pH value for E. vermicola was tested. The diameter and dry mass of E. vermicola in PDA medium were increased greatly at pH 5 after 15 days, while the diameter and dry mass of E. vermicola were inhibited at pH 9 and 10. The sporulation of E. vermicola in PDA or PDB medium was highest at pH 6, 2.0 × 10 7 and 1.4 × 10 7 CFU/ml, respectively. The proportion of lunate conidia in PDA medium was highest at pH 4, while the proportion of lunate conidia in PDB medium was highest at pH 7.


INTRODUCTION
Esteya vermicola, an endoparasitic fungus of pinewood nematode, exhibits great potential as a biological agent against pinewood nematode.It produces two kinds of conidia, bacilloid and lunate conidia.Bacilloid conidia do not adhere well to nematodes whereas lunate conidia can adhere and infect nematodes by cuticle penetration, immobilization and digestion of the internal contents (Liou et al., 1999).E. vermicola showed good results in conptrolling the pinewood nematode by inoculating conidia suspensions into red pine tree (pinus densiflora) logs which were killed by pinewood nematodes, or spraying conidial suspensions onto pine seedlings under greenhouse conditions (Wang et al., 2010).The conidia were applied under the condition of different pH values, so the range of pH values for E. vermicola growing and the optimization of pH value for mass production should be considered.
It was reported that a medium having pH values between 5 and 6 at the time of inoculation is suitable for most fungi, because fungi generally tolerate more acid than alkali (Lilly and Barnett, 1951).Colletotrichum gloeosporioides isolates grow well at pH 5 while sporulation is better at pH 6 (Kumara and Rawal, 2008).Similar observations are also reported by some other authors with different species of Colletotrichum (Ramakrishnan, 1941;Sood, 2011).Mycelia of Coniothyrium minitans at pH 6 have a higher productivity of conidia production than those grown at pH 4 (Shi, 2008).For Ceratocystis paradoxa, the PDA recorded highest diameter at pH 6.5 whereas sporulation intensity was highest at pH 7.0 (Yadahalli, 2007).

Fungal strain
E. vermicola CNU 120806 was obtained from the Agriculture Bioscience Biotech Center, Chungnam National University, Korea.
It was maintained on potato dextrose agar (PDA) (Acumedia, USA) slants at 4°C and cultured on PDA plates at 26°C.

Culture media
A 4.8 g of potato dextrose broth powder was added into a flask with 200 ml distilled water and was mixed by a glass rod.The pH value of the PDB solution was adjusted to 4, 5, 6, 7, 8, 9 and 10 with HCl solution (11.7%) and NaOH solution (5 M/L).For solid medium, agar powder was added at the level of 3.5% to the PDB solution at pH 2, and at the level of 3% to the PDB solution at pH 3 and 4, and at the level of 2% at pH 5, 6, 7, 8, 9 and 10.For liquid culture medium, no agar was added to the PDB broth.After PDA medium were autoclaved, the PDA medium was poured into Petri dishes.
Cellophane was cut to similar size of Petri dish, transferred into flask with water and autoclaved.In order to separate fungal colony from culture medium, the sterile cellophane discs were placed onto the PDA medium in Petri dishes before inoculation.A 5-mm fungal disc was taken from the periphery of 7-day-culture E. vermicola colony and transferred to the center of the PDA medium in each Petri dish, and cultured in an incubator for 15 days at 26°C.For liquid medium, 3 discs from the edge of 7-day-old pure culture (1 cm in diameter) were added to the PDB broth and cultured for 7 days at 26°C.

Growth rate, sporulation and proportion of lunate conidia
The growth rate of E. vermicola in PDB medium in different pH values was evaluated after 7 days, and the growth state in PDA medium in different pH values was evaluated after 15 days.For PDB medium in different pH values, the mycelium in liquid culture medium was filtrated by filter paper and was dried in oven at 60°C for 2 days.The suspension of PDB medium was oscillated using an oscillator (Vortex genie, USA) at a speed of 600 oscillations per minute with 0.5% (v/v) Tween 80 (Sumchun, Korea) and several glass beads.Concentration of conidia was determined using a hemocytometer (Marienfeld, Germany).The size of two conidia which were produced from E. vermicola was quite different.One was lunate which can adhere, infect and kill nematode, the other was bacilloid.The lunate conidia were counted and the proportion of lunate conidia was measured.
Diameters of colonies were measured in PDA medium in different pH values.Some colonies which were cultured in PDA medium were isolated from cellophane and transferred to an oven at 60°C for 2 days.Furthermore, other colonies which were cultured in PDA medium were transferred into 50 ml tubes with 0.5% (v/v) Tween 80 and several glass beads.To dislodge conidia from conidiophores, the tubes were oscillated using an oscillator at a speed of 600 oscillations per minute.Concentration of conidia was determined using a hemocytometer.The lunate conidia were counted and the proportion of lunate conidia was measured.

Data analysis
All experiments were conducted in three replications and repeated 3 times, and the data were presented as means and standard deviations.The effects of pH value on growth, sporulation and proportion of lunate conidia were compared and analyzed statistically.The significance of differences was determined by one-way analysis of variance (ANOVA) with Tukey's multiple comparison module of the Minitab statistical software, version 13.30 (Minitab Inc., State College, PA, USA) and differences with P values of <0.05 were considered statistically significant.

Effects of different pH values on growth rate
Effects of different pH values on diameter and dry mass of E. vermicola in PDA and PDB medium are shown in Table 1.The pH range of E. vermicola for growth varied between 4 and 10 in PDA medium, and varied between 4 and 9 in PDB medium.The diameter of E. vermicola in PDA medium was increased greatly at pH 5, 6 and 7 after 15 days, while the diameter of E. vermicola was inhibited greatly at pH 10.Effects of different pH values on diameter of E. vermicola in PDA medium showed positive correlation to dry mass.Dry mass was increased greatly in PDA medium at pH 5 and 6, 4-fold more than that at pH 10.In PDB medium, the dry mass was enhanced at pH 5 and 6 after 7 days, while the growth of dry mass was inhibited greatly at pH 9 and 10.
E. vermicola grew better in weak acid environment than in weak alkaline environment.Moreover, E. vermicola could not grow in PDA medium below pH 3 and above pH 11, and it could not grow below pH 3 and above pH 10 in PDB medium.That may be because the strong acid or strong base destroyed the DNA of chromosome and protein, and inhibited conidia growth.

Effects of different pH values on sporulation
Effect of different pH values on sporulation of E. vermicola in PDA medium is shown in Figure 1.The sporulation of E. vermicola was enhanced greatly at pH 6, 3.3 × 10 7 CFU, 3-fold more than that at pH 9 and 10.The sporulation was inhibited gradually above pH 7.
Therefore, E. vermicola produced more conidia in weak acid environment than that in weak alkaline environment.
Effect of different pH values on sporulation of E. vermicola in PDB medium is shown in Figure 2. The sporulation of E. vermicola was enhanced greatly at pH 5 and 6 in PDB medium, while the sporulation was inhibited gradually above pH 7. The growth rate was highest at pH 5 in PDA or PDB medium, while the sporulation was highest at pH 6 in PDA or PDB medium.That may be because E. vermicola could grow more quickly and produce more mycelia at pH 5 than that at pH 6, however conidiophores could produce more lunate and bacilloid conidia at pH 6 than that at pH 5. Sood (2011) got the similar result that growth of Aspergilli umbrosus in Asthana and Hawker's, and Currie's medium was optimum at pH 5 while sporulation and secondary metabolite production were moderate.

Effects of different pH values on proportion of lunate conidia
Effects of different pH values on the proportion of lunate conidia in PDA or PDB medium are shown in Figure 3.The proportions of lunate conidia in PDA or in PDB medium were higher at pH range between 4 and 7 than that at pH range between 8 and 10.The proportion of lunate conidia in PDA medium was highest at pH 4, 4-fold higher than that at pH 8.Moreover, the proportion of lunate conidia in PDB medium was highest at pH 7, 11fold higher than that at pH 8.In this study, the proportion of lunate conidia in PDB medium was higher than in PDA medium.It was reported that in vitro on enriched media, the hyphae produced primarily the cylindrical, elongate, subulate multiseptate, simple or branched conidiophores and conidiogenous cells bearing cylindrical to bacilloid, not lunate conidia of E. vermicola (Liou, 1999).The solid PDA medium con-tains more nutrition than the liquid PDB medium.Moreover, it can provide more oxygen for fungal growth.The PDA medium produced less lunate conidia, therefore, it had lower proportion of lunate conidia than that in PDB medium.
In conclusion, the growth rate, sporulation and proportion of lunate conidia in PDA and PDB medium were increased greatly in pH values between 5 and 7.The growth rate was highest at pH 5 in PDA and PDB medium, and the sporulation of E. vermicola was highest at pH 6 in PDA and PDB medium.The proportion of lunate conidia in PDA medium was highest at pH 4, and the proportion of lunate conidia in PDB medium was highest at pH 7.This study may provide proper pH value for mass culture and application of E. vermicola to control pine wilt disease.

Figure 1 .Figure 2 .
Figure 1.Effect of different pH values on sporulation of E. vermicola CNU 120806 in potato dextrose agar medium cultured at 26°C for 15 days.Letters above similar bars indicate significant differences at P<0.05 for Turkey's test.

Figure 3 .
Figure 3.Effect of different pH values on proportion of lunate conidia of E. vermicola CNU 120806 in potato dextrose agar and potato dextrose broth medium.Letters above similar bars indicate significant differences at P<0.05 for Turkey's test.

Table 1 .
Effect of different pH values on diameter and dry mass of E. vermicola CNU 120806 in potato dextrose agar medium and potato dextrose broth medium.