cordyceps militaris

  ʾ 7   1 2552

Kamphaengsean Acad. J. Vol. 7, No. 1, 2009, Pages 1-9

    Cordyceps spp. F F     x 108 Growth of Cordyceps spp. on the Pupae of Thai Silkworm, Bombyx mori  Nanglai X 108      1*    F F  F F1 Siripuk Suraporn 1*  and Wanwisa Siriwattanametanon 1

ABSTRACT

The growth of  Cordyceps spp. and their anamorphs on the pupae of the Thai hybrid silkworm, Bombyx mori  Nanglai x 108 was investigated. Six Cordyceps strains; Cordyceps pseudomilitaris (945.2), C. pseudomilitaris ( 951.1), C. khaoyaiensis (885.1), C. khaoyaiensis (5658), Cordyceps sp. (5273), Cordyceps sp. (4653), and the anamorph of  Cordyceps; Paecilomyces tenuipes (JapanRN474) were used as the test subjects. The entomopathogenic fungi, Beauveria bassiana (10611) was used as a positive control. The pupae (7 days old) were divided divided into two groups, the the first group were killed by incubation at 70 C for 2 hours and the second group were kept alive. Three methods of  fungal inoculation of the pupae were used; direct injection into the pupae of 0.1 ml of 10 8 spores/ml suspended spores, application of 0.1 ml of 10 8 spores/ml suspended spores, and direct mycelial inoculation over the pupal surface. Inoculated pupae were incubated at 24-25 C with 80-90% humidity for the fungal growth of 14 days. The most effective inoculation methods were injection of suspended spores and direct mycelial inoculation. Fungal mycelial growth on incubated pupae could be obviously seen within 2 days and 3-4 days after inoculation, respectively. The killed pupae were more prone to penetration by Cordyceps spp. than live pupae. However, only P. tenuipes produced elongated fruiting body (stroma) pertruding from the cadaver of pupae. Examination of the fungal structures on the silkworm pupae revealed that they were of the Cordyceps spp. that were initially inoculated and were of the following characters; hyphae were we re septated and globose shaped asexual spores were of various sizes. B. bassiana (10611) also produced septate hyphae and clusters of conidiogenous cells with long and narrow rachis and hyaline globose conidia. ˚

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Key words: Cordyceps spp., Beauveria bassiana, Thai silkworm, Nanglaix108

1*

         .  44150 Department of Biology, Faculty of Science, Mahasarakham University 44150, Thailand. *Corresponding author: Tel/Fax 0-4375-4245. Email address: [email protected]

2

 ʾ 7  1 2552



   Cordyceps 6    Cordyceps pseudomilitaris (945.2), C. pseudomilitari s (951.1), C. khaoyaiensis (885.1), C. khaoyaiensis (5658), Cordyceps sp. (5273)  Cordyceps sp. (4653)  Paecilomyces tenuipes (Japan- RN 474; anamorph  Cordyceps)   x108    Beauveria bassiana (10611; anamorph  Cordyceps)) ˈ    7  ˈ 2        70   2        ()    3    tween 80 (108 /)   (108 /)          3     24-25     80-90 ˈ 14                   2   3-4                    P. tenuipes (Japan- RN 474)     stroma       ˈ         Cordyceps spp.    6    P. tenuipes (Japan- RN 474)    (septate hypha)  conidia   conidia ˈ    B. bassiana (10611)  conidia    conidiogenous cell   ˈ      : Cordyceps spp. Beauveria bassiana  x108 

    Cordyceps ˈ         Ascomycetes  Hypocreales    Clavicipitaceae (Hawksworth et al ., 1995)                   Diptera, Hymenoptera, Isoptera  Lepidoptera       Cordyceps  stroma      (, 2538)    Cordyceps  cordycepic acid  cordycepin  ˈ isomer   quinic acid (Isaka et al ., 2000)            Cordyceps     ˈ     

                   ˈ        Cordyceps sinensis     ( Lepidoptera) ˈ         ( , 2544)   ˁ                       Paecilomyces japonica  ˈ              ˈ      (Lee et al., 2001)        Cordyceps ˈ  Cordyceps pseudomilitaris  ˈ           

 ʾ 7  1 2552

     human immunodeficiency virus-1 reverse transcriptase (anti-HIV-1 RT) ( , 2544)             ˈ           (, 2540;   , 2540)       ˈ    Cordyceps  ˈ              Cordyceps spp.  P. tenuipes    ˈ           ˈ    F 

  x108 ˆˈ18  /   10  ˆˈ     2        5      33-37       7      70  ˈ  2     ˀ  ˊ  ˈ           70   2          (ˈ  )    F 

                         (.)

3

 Cordyceps spp.  6    (945.2), C. Cordyceps pseudomilitaris pseudomilitaris (951.1), C. khaoyaiensis (885.1), C. khaoyaiensis (5658), Cordyceps sp. (5273), Cordyceps sp. (4653)     ˈ  anamorph  Cordyceps  Paecilomyces tenuipes ( Japan- RN 474)        7            potato dextrose agar (PDA)             7      Beauveria bassiana (anamorph  Cordyceps)  ˈ   ˈ          (inocula)    2    (mycelial inoculum)   (suspended spores)  108 /   tween 80 (0.1%)                       potato dextrose agar (PDA)   250              20       ˈ 14               (suspended spores)  tween 80  1   80           loop        haemacytometer   108 /        (inoculate)     10 /   1  /   (  )        ˈ  ˈ  14          ˈ 3                 

4

 ʾ 7  1 2552

(    )        24-25                 10 /    1     1                          0.1                                 ˈ 3     24-25          0.1         10 /1     1                           ˈ 3     24-25     

      ˈ     7         potato dextrose agar      fruiting structures                 

  F   F 

      7    C. pseudomilitaris (945.2), C. pseudomilitaris (951.1), C. khaoyaiensis (885.1), C. khaoyaiensis (5658), Cordyceps sp. (5273), Cordyceps sp. (4653)  P. tenuipes (Japan - RN 474)  B. bassiana (10611) ˈ                70   2                  24-25        80 -90     (Figure 1)   tween 80 ˈ            (Figure 2A and 2B)  C. pseudomilitaris (945.2 ), P. tenuipes (Japan-RN 474), C. pseudomilitaris (951.1), Cordyceps sp. (5273)  Cordyceps sp. (4653)     95, 93, 92, 91  90  (Table 1)              C. pseudomilitaris (945.2 )          2           B. bassiana (10611)         95           P. tenuipes ( Japan-RN 474)      stroma     45  (Figure 2C)

5

 ʾ 7  1 2552

igure 1 Early fungal growth on silkworm pupae, Bombyx mori in test tubes at room temperature

 A

B

C

Figure 2 The growth of Paecilomyces tenuipes on live silkworm pupae, Bombyx mori 

(A),

heat killed pupae (B) and

incubation.

the stroma of  P . tenuipes on silkworm pupae after 45 days

6

 ʾ 7  1 2552

Table 1 Growth of Cordyceps strains, Paecilomyces tenuipes and the entomopathogenic

fungi; Beauverai bassina on the pupae of Thai silkworm, Bombyx mori  Nanglai X108 using injection of suspended spores, application of suspended spores, and mycelial inoculation methods. Fungal strain

Injection of suspended spores Application of suspended spores Mycelial inoculation Heat killed pupae live pupae Heat killed pupae live pupae Heat killed pupae live pupae level of  level of  level of  level of  level of  level of  inoculation inoculation inoculation inoculation inoculation Inoculation mycelial mycelial mycelial mycelial mycelial mycelial (%) (%) (%) (%) (%) (%) production production production production production production

C. pseudomilitaris

(945.2)

95

+++

90

+++

90

+++

90

++

93

+++

93

++

92

+++

91

+++

81

++

79

++

90

+++

90

+++

89

+++

87

+++

79

+++

76

++

90

+++

87

+++

85

+++

85

++

77

++

77

+++

88

+++

81

+++

91

+++

90

++

86

+++

84

++

90

+++

90

++

90

+++

87

+++

77

++

72

++

89

++

90

++

93

+++

89

+++

90

++

78

++

93

+++

91

+++

95

+++

93

+++

88

+++

83

++

94

+++

90

++

C. pseudomilitaris

(951.1) C. khaoyaiensis

(885.1) C. khaoyaiensis

(5658) Cordyceps sp.

(5273) Cordyceps sp.

(4653) P. tenuipes

(Japan - RN 474) B. bassiana

(10611)

Note : +++ = very good, ++ = fair, and + = poor 

                5            90   Cordyceps sp. (5273), C. pseudomilitaris (951.1), C. khaoyaiensis (885.1), C. khaoyaiensis (5658)  Cordyceps sp. (4653)   86, 81, 79, 77  77   C. pseudomilitaris (945.2 )  P. tenuipes ( Japan-RN 474)  90             C. pseudomilitaris (945.2 )   

  B. bassiana (10611)          88              3-4                    P. tenuipes (Japan-RN 474), C. pseudomilitaris (945.2 ), C. pseudomilitaris (951.1), Cordyceps sp. (4653)  Cordyceps sp. (5273)    94, 93, 90, 90  90   C. pseudomilitaris (945.2 )    

 ʾ 7  1 2552

                         Cordyceps sp. (4653)  B. bassiana (10611)     94     stroma     F    F F 

      7             C. pseudomilitaris (945.2 ), P. tenuipes (Japan - RN 474), C. pseudomilitaris (951.1), C. khaoyaiensis (5658), C. pseudomilitaris (951.1), Cordyceps sp.(4653), Cordyceps sp (5273)     PDA           (Figure 3A)        (Figure 3B) conidia   ˈ    conidiophore       ˁ               B. bassiana     PDA     (Figure 4A)                     conidia      conidiogenous cell    (Figure 4B) 

        Cordyceps spp. 6    P. tenuipes (Japan-RN 474)                   B. bassaina (10611) ˈ     

7

ˈ         2            3-4   5            C. pseudomilitaris (945.2), P. tenuipes (Japan-RN 474)  C. pseudomilitaris (951.1)           95, 93  92      B. bassiana (10611)  95            70   2                            chitinase                        24 - 25       80-90              C. pseudomilitaris (945.2), P. tenuipes (Japan-RN 474)  C. pseudomilitaris (951.1)          ˈ                 ˆ       Paecilomyces japonica      ˈˆ           25     95     24L  24D (Lee et al ., 2001)      Cordyceps  P. tenuipes (Japan-RN 474)   (septate

8

 ʾ 7  1 2552

hypha)  conidia    conidia  ˈ     B. bassiana (10611)  conidia    conidiogenous cell    ˈ           ˈ     ˆ         Cordyceps  P. tenuipes (Japan-RN 474)           

                (2544)                      ˈ  ˈ          

A

B

igure 3 Hyphal growth of  Cordyceps spp. on Potato Dextrose Agar (A) and micrograph showing

conidia of Cordyceps spp. under light microscope (40X) (B).

A B igure 4 Hyphal growth of  Beauveria bassiana on Potato Dextrose Agar (A) and micrograph showing conidia of Beauveria bassiana under light microscope (40X) (B).

 ʾ 7  1 2552



                                           ..        .            F

     . 2538.                               ..     . 2540.  ˆ                              , .  . 2544.                    , .        . 2540.             Received 3 July 2008 Accepted 30 April 2009

9

  , .   . 2544.                                     , . Hawksworth, D.L., P.M. Kirk, B.C. Sutton. 1995. Ainsworth and Bisbys Dictionary of the Fungi. CAB International, Wallingford UK. 616 p. Isaka M., J. Punya, Y.Lertwerawat, M.Tanticharoen, and Y. Theptaranonth 2000. Antiplasmodial compounds from wood-decayed fungus  Xylaraia. BCC 1067. Plant Med. 66: 473-475. Lee E.H, S. P. Nam, B. Sang, and L . Park. 2001. Effects of Photoperiods on the Growth of the Entomopathogenic Fungi, Paecilomyces japonica, During the Production of the Silkwormdongchunghacho, Silkworm Vegetable Wasp and Plant Worm. Int. J. Indust. Entomology. Vol. 2 (1): 83-86.