Optimization of nutritional requirements for mycelial growth and sporulation of entomogenous fungus Aschersonia aleyrodis Webber.

The objective of the present study was to investigate the optimal nutritional requirements for mycelial growth and sporulation of entomopathogenic fungus Aschersonia aleyrodis Webber by orthogonal layout methods. Herein the order of effects of nutrient components on mycelial growth was tryptone > Ca(2+) > soluble starch > folacin, corresponding to the following optimal concentrations: 1.58% Soluble Starch, 3.16% Tryptone, 0.2 mmol l(-1) Ca(2+) and 0.005% Folacin. The optimal concentration of each factors for sporulation was 1.16% lactose, 0.394% tryptone, 0.4 mmol l(-1) Fe(2+) and 0.00125% VB1, and the effects of medium components on sporulation were found to be in the order lactose > VB1 > Fe(2+) > tryptone. Under the optimal culture conditions, the maximum production of mycelial growth achieved 20.05 g l(-1) after 7 days of fermentation, while the maximum spore yield reached 5.23 ×10(10) spores l(-1) after 22 days of cultivation. This is the first report on optimization of nutritional requirements and design of simplified semi-synthetic media for mycelial growth and sporulation of A. aleyrodis.

INTRODUCTION

Aschersonia aleyrodis Webber belongs to Ascomycota; Pezizomycotina; Sordariomycetes; Hypocreomycetidae; Hypocreales; Clavicipitaceae; mitosporic Clavicipitaceae; Aschersonia. This fungus was firstly used for controlling whiteflies in citrus groves in Florida in the early 1900s (1), where a combination of predators, parasitoids with A. aleyrodis successfully controlled the citrus whiteflies Dialeurodes citri and D. citrifolii for decades (12). Previous researches have indicated that A. aleyrodis is a promising whitefly control agent because of its long persistence on leaf surfaces (6,13) and its compatibility with Encarsia formosa (7,8). Along with A. aleyrodis several other species of the genus Aschersonia have been reported on whitefly species all over the world (11,14,15). This genus is known to cause severe epizootics in whitefly (Aleyrodidae) and scale insects (Coccidae) in the tropics and subtropics (4). Epizootics of A. aleyrodis have been reported from Bemisia tabaci populations; however, most microbial control efforts with this fungus have targeted the greenhouse whitefly, Trialeurodes vaporariorum (9).

Mycelial growth and sporulation on artificial media are important biological characteristics of fungi, including A. aleyrodis. And the nutritional requirements can have a profound effect on culture growth, conidiation and morphology in insect pathogenic fungi. However, to the best of our knowledge, the nutritional requirements of A. aleyrodis have not been demonstrated so far. Also, there has been no suitable medium for the cultivation of A. aleyrodis till now, and this fungal pathogen does not grow well in any known medium. In order to further facilitate the physiological study of this fungus and its application in biocontrol more information is required. The first step is to increase the biomass and sporulation in an artificial culture. Generally, the media used in large scale fermentations of entomopathogenic fungi contain raw natural materials, such as corn steep powder, potato, soya bean, etc., and the chemical compounds of the media were not quantifiable. Although these raw components of media may have the advantage of low cost, it may not be possible to control the quality of the A. aleyrodis mycelial product and sporulation for the heterogeneity or batch variation of the natural components. Because of the unquantified chemical constituents, the study on physiological and biochemical characters of A. aleyrodis can not be performed in these media. To further study the physiology of A. aleyrodis and improve mycelial growth and sporulation of A. aleyrodis, as well as to quantify the compounds used in cultivation media, synthetic or semi-synthetic media are required.

In a previous experiment, 12 carbon sources (dextrose, sucrose, D-xylose, lactose, maltose, D-fructose, soluble starch, inositol, mannitol, D-galactose, α-Lactose, sorbitol), 10 nitrogen sources ((NH4)2SO4, NaNO3, NH4NO3, urea, yeast powder, tryptone, casein, peptone, beef extract, yeast extract), 6 metal ions (Cu2+, Mn2+, Zn2+, Fe2+, Fe3+,Ca2+) and 6 vitamins (VB1, VB2, VB5, VC, VB6, folacin) were studied using one-factor-at-a-time method in order to evaluate the effects of various C and N sources, metal ions and vitamins on mycelial growth and sporulation of this fungus. For the C source test, 7 g of C l-1 each carbohydrate was added individually to the basal medium (dextrose 2% (w/v), (NH4)2SO4 0.2% (w/v), KCl 0.05% (w/v), MgSO4 0.05% (w/v), KH2PO4 0.05% (w/v), Na2HPO4 0.065% (w/v) and distilled water) to replace dextrose and a basal medium free of any C was served as a control. For the N source test, 0.5 g of N l-1 each N compound was added respectively to the basal medium to replace (NH4)2SO4 and a basal medium without N acted as a control. For the metal ions test, 4×10–4 mol each metal ion was added to l L basal medium, respectively, and basal medium was served as a control. For vitamin test, 100 mg each vitamin was added to l L basal medium, respectively, and basal medium was served as a control. According to the results of previous study, we found that the best nutrient components for the mycelial growth of this fungus included soluble starch, tryptone, Ca2+ and folacin, while the best nutrient components for sporulation included lactose, tryptone, Fe2+ and VB1 (18). After the best nutrient components for this fungus were screened out, the effective concentrations of these components were further investigated by the concentration gradient experiments of each component. The experiment procedures are similar to that of one-factor-at-a-time experiments described above, and the 7 different concentrations of each best component replaced the corresponding C or N source of basal medium or added into the basal medium individually (data not shown). Based on the results of these experiments 3 effective concentrations of each component listed in Table 1 and 2 respectively were chosen for optimization experiments.

Table 1

L9 (34) orthogonal design on optimization of culture medium for mycelial growth.

Factors	Soluble starch (SS)a (% w/v)	Tryptone (TR) (% w/v)	Ca2+(mmol l-1)	Folacin (FO) (% w/v)
Level 1	0.79	0.79	0.2	0.005
Level 2	1.58	1.58	0.4	0.01
Level 3	3.16	3.16	0.8	0.02

SS, TR and FO represent factors of soluble starch, tryptone and folacin respectively.

Table 2

L9 (34) orthogonal design on optimization of culture medium for sporulation.

Factors	Lactose (LA)a (% w/v)	Tryptone (TR) (% w/v)	Fe2+(mmol l−1)	Vb1 (FO) (% w/v)
Level 1	0.29	0.197	0.2	0.00125
Level 2	0.58	0.394	0.4	0.0025
Level 3	1.16	0.788	0.8	0.005

LA and TR represent factors of lactose and tryptone respectively.

In this paper, the optimal nutrient requirements were studied by orthogonal matrix method on the basis of previous work in order to get more information of the fungus.

MATERIALS AND METHODS

Fungal isolates

Based on previous bioassays against whitefly using 40 strains of A. aleyrodis isolated by our laboratory, strain labelled JO009 was selected for this study. The strain was originally isolated from a citrus whitefly D. citri from a citrus-growing orchard in Fujian, China, in 2000. A single-spore isolate of JO009 was stored in 20% glycerol at -80ºC and transferred once every 4 months. Cultures were grown on potato dextrose agar (PDA) at 25ºC with a daily cycle consisting of 15 h of light and 9 h of darkness. The strain was inoculated twice in sweet-potato whitefly (B. tabaci) before using in the assay.

Inoculum preparation

Spores of this fungus were harvested from potato dextrose agar (PDA) slant cultures after incubation at 25±1ºC for 2 weeks, then transferred into 50 ml sterile solution containing 0.05% Tween 80, and stirred with a magnetic stirrer for 30 min to dislodge and suspend the spores and finally passed through three layers gauze (or 100-mesh sieve). Spores were counted by using a haemacytometer and adjusted with sterile 0.05% Tween 80 solution to produce final concentrations of 1.66×107 spores ml-1.

Orthogonal layout

Based on the results of our previous work, the screened nutrient components which are most suitable for mycelial growth and sporulation of A. aleyrodis and the 3 effective concentrations of each component were further optimized using the orthogonal layout L9(34). The levels of components of the media for mycelial growth and sporulation are shown in Table 1 and 2, respectively.

Besides the screened C sources, N sources, metal ions and vitamins listed in Table 1 and 2, media used in present experiment also contained 0.05% (w/v) KCl, 0.05% (w/v) MgSO4, 0.05% (w/v) KH2PO4, 0.065% (w/v) Na2HPO4 and 2%-5% agar (agar is not added in media for mycelial growth). Metal ions were provided with ZnSO47H2O, FeSO47H2O and CaCl2 respectively. Metal ions and VB1 were sterilized by filtering with a 0.2-µm aperture filter and were added to the sterile medium under sterile conditions. Other vitamins were added to the medium prior to heat sterilization (2,16).

Analytical methods

For mycelial growth test, 200 µl spore inoculum was added into 500 ml flasks each containing 200 ml of liquid medium and then cultured in a rotatory shaker at 150 rpm and 25±1ºC. After 7 d, the mycelial biomasses were harvested by vacuum filtration with pre-dried and weighed filter papers and then mycelium was dried at 80ºC for 24 h. The growth was estimated by the dry weight of mycelium. Each treatment was repeated three times.

For sporulation test, 200 µl spore inoculum was transferred onto the center of each petri plate (9 cm in diameter) containing 15 ml solid media, sealed with parafilm, and then placed in an incubator at 74% relative humidity (RH) and 25±1ºC. After 22 d, spores were washed with 10 ml 0.05% Tween 80 solution and stirred on a magnetic stirrer for 30 min and then passed through three layers gauze (or 100-mesh sieve). The number of spores was determined using a hemocytometer with the aid of a microscope (magnification × 300). Four duplications were made for each treatment.

Statistical analysis

Data of orthogonal tests were analyzed using variance analysis. Differences of F < 0.05 or F > 0.01 were considered different significant levels.

RESULTS

Mycelial growth test

Factors and levels of orthogonal layout L9(34) for mycelial growth are shown in Table 1, and the conditions for each experimental group are listed in Table 3 including the results in the last column. As shown in Table 3, the maximum production of mycelia (17.75 g l-1) occurred in the sixth experimental group whose condition was: SS-2, TR-3, Ca2+-1 and FO-2, namely soluble starch (1.58%), tryptone (3.16%), Ca2+ (0.2 mmol l-1) and folacin (0.01%), respectively. According to the magnitude order of R (maximum difference) in the last row of Table 3, the order of the effect of all factors on mycelial growth could be determined. The order of effects of factors on mycelial growth was tryptone (66.99) > Ca2+ (32.31) > soluble starch (29.72) > folacin (15.53). This result pointed out that the effect of tryptone was more important than that of other nutrients. The result that tryptone had the largest F ratio (48.85) (Table 4) also demonstrated thus. In addition, the variance analysis shown in Table 4 indicated that those factors affected the mycelial growth of A. aleyrodis significantly except folacin whose F ratio (2.52) was smaller than the value of F005 (2,18).

Table 3

The results of L9 (34) orthogonal test of mycelial growth.

Experimental group	SSa	TR	Ca 2+	FO	Mycelial dry weight (g l-1)b
1	1	1	1	1	7.30±0.54
2	1	2	2	2	4.32±1.19
3	1	3	3	3	10.65±1.81
4	2	1	2	3	5.29±1.08
5	2	2	3	1	9.13±3.83
6	2	3	1	2	17.75±1.58
7	3	1	3	2	6.84±1.73
8	3	2	1	3	8.69±1.51
9	3	3	2	1	13.37±2.19
K1c	66.79	58.29	101.24	89.39
K2	96.51	66.41	68.93	86.73
K3	86.68	125.28	79.81	73.86
Rd	29.72	66.99	32.31	15.53

SS, TR and FO represent factors of soluble starch, tryptone and folacin respectively.

Values are mean ± SD of triple determinations.

K1, K2 and K3 are the total content of mycelial dry weight from level 1, level 2 and level 3 respectively.

R is the maximum of K1, K2 and K3 minus the minimum of K1, K2 and K3, respectively.

Table 4

The variance analysis of L9 (34) orthogonal test on mycelial growth.

Variance source	F ratiob	Significance levelc
SSa	8.38	**
TR	48.85	**
Ca2+	9.88	**
FO	2.52

SS, TR and FO represent soluble starch, tryptone and folacin respectively.

The degrees of freedom for all factors are all 2. And the degree of freedom for error is 18.

F0.05(2, 18) = 3.55; F0.01(2, 18) = 6.01; * F0.05 F0.01.

Optimal level of each medium ingredient for the mycelial growth of A. aleyrodis was determined in terms of the maximum K value of each column in Table 3. The optimum compositions were SS-2, TR-3, Ca2+-1 and FO-1 (1.58% soluble starch, 3.16% tryptone, 0.2 mmol l-1 Ca2+, 0.005% folacin). To confirm these data, experiments were carried out using these nutrient concentrations and 20.05 g l-1 of mycelial biomass was obtained. This implied that the selected conditions were the most suitable in practice.

Sporulation test

According to the four factors and three levels of the orthogonal layout for sporulation (Table 2), the conditions for each experimental group were listed in Table 5 with the results concluded in the last column. From the Table 5, it could be found that the highest mean yield of spore (4.79×1010spores l-1) by A. aleyrodis was reached at the ninth experimental group and the corresponding condition was: LA-3, TR-3, Fe2+-2 and VB1-1, namely 1.16% lactose, 0.788% tryptone, 0.4 mmol l-1 Fe2+ and 0.00125% VB1, respectively. Based on the magnitude order of R value shown in the last row of Table 5, the order of effects of all factors on sporulation of A. aleyrodis was estimated respectively. The order of effects of all factors on sporulation was lactose (26.50) > VB1 (16.35) > Fe2+ (15.17)> tryptone (4.96). This result indicated that the effect of lactose was more important than that of other factors. To test the effects of the four factors, the variance analysis was used and shown in Table 6. Three factors, including lactose, Fe2+ and VB1, whose F ratios were all larger than the value of F (2,27) when the P value is 0.01, had extremely significant effects on sporulation of A. aleyrodis, especially lactose whose F ratio (50.16) is the largest of all, whereas tryptone (F ratio=1.54) did not display significant influence on sporulation of this fungus.

Table 5

The results of L9 (34) orthogonal test of sporulation.

Experimental group	LAa	TR	Fe2+	Vb1	Sporulation (×1010spores 1−1)b
1	1	1	1	1	1.67±0.25
2	1	2	2	2	1.42±0.23
3	1	3	3	3	0.23±0.09
4	2	1	2	3	1.06±0.23
5	2	2	3	1	1.40±0.86
6	2	3	1	2	0.52±0.24
7	3	1	3	2	1.83±0.47
8	3	2	1	3	2.98±0.51
9	3	3	2	1	4.79±1.22
K1c	13.26	18.23	20.67	31.41
K2	11.90	23.19	29.03	15.06
K3	38.40	22.14	13.86	17.09
Rd	26.50	4.96	15.17	16.35

LA and TR represent factors of lactose and tryptone respectively.

Values are mean ± SD of tetrad determinations.

K1, K2 and K3 are the total content of spore yield from level 1, level 2 and level 3 respectively.

R is the maximum of K1, K2 and K3 minus the minimum of K1, K2 and K3, respectively.

Table 6

The variance analysis of L9 (34) orthogonal test on sporulation.

Variance source	F ratiob	Significance levelc
LAa	50.16	**
TR	1.54
Fe2+	13.00	**
VB1	17.89	**

LA and TR represent lactose and tryptone respectively.

The degrees of freedom for all factors are all 2. And the degree of freedom for error is 27.

F0.05(2, 27) = 3.35; F0.01(2, 27) = 5.49; * F0.05 F0.01.

In terms of the maximum K value of each column in Table 5, optimal level of each medium ingredient for the sporulation was LA-3 / TR-2 / Fe2+-2 / VB1–1. To obtain a high spore yield, the optimum composition should be 1.16% lactose, 0.394% tryptone, 0.4 mmol l-1 Fe2+ and 0.00125% VB1. To confirm these data, experiments were carried out using these nutrient concentrations and 5.23×1010spores l-1 of spore yield were obtained. And the germination rate of spores on water agar plate achieved 100% after incubated in an incubator at 74% RH and 25±1ºC for 16h.

DISCUSSION

Culture media has been shown to influence the germination, mycelial growth, sporulation and virulence of fungi employed as mycoinsecticides (5,10). In previous work, we firstly reported effects of 12 carbon sources, 11 nitrogen sources, 6 metal ions and 6 vitamins on growth and development of A. aleyrodis (18). According to the results of our previous study, optimization of nutritional requirements and design of simplified semi-synthetic media for mycelial growth and sporulation of A. aleyrodis were firstly reported in this paper.

The conventional variation of one-factor-at-a-time approach of optimization is not only time-consuming but often incapable of reaching the true optimum due especially to the interactive effects among factors. Orthogonal layout can give effective responses because of the suitable design of factor. In comparison with the full-factors experimental design, orthogonal design can reduce experimental difficulties and reveal the interactions of factors (3,17). In the present experiment, the orthogonal matrix method has helped us to understand the nutritional requirements of mycelial growth and sporulation of A. aleyrodis and obtain the optimal media including the ratio of each medium component.

By using chemically synthetic media, the effect of medium components on nutritional requirements of mycelial growth and sporulation of A. aleyrodis can be studied in detail. Moreover, process consistency is enhanced because the chemically semi-defined media inherently supports a more reproducible process. The optimized media designed in this study is useful for the investigation of fermentation kinetics of mycelial growth in A. aleyrodis and can also be used to produce mycelial and spore products of consistent quality. There are many factors (i.e. temperature, humidity, pH, etc.) which can influence A. aleyrodis, and there must be interactions among these factors. Basic and essential information for the mass production of A. aleyrodis in culture allows for some insights into the physiology of this fungus. It is necessary to further research the other factors and optimize the fermentation conditions in a fermenter using complex media to achieve the demands of large-scale mycelial production and sporulation, which are an ongoing project in this laboratory.