Òî
ŢƟŚƿŹŵ í ì æèîæ ƁźƿŸě
çç æç æèîæ
ƶƯŚƳ ƵźƄůƲưŬƳřƽ
ƱřźƿřƾſŚƴƃ
ëì êî æ èè æèîç
ſŹźŝ ƾ ŝǀ ƱĥƱŚ ŚƷ ƽ
CHit1
ƹ
CHit2
ġŹŚƣŹŵ
Beauveria bassiana
ŻřŹŚºěƪºƯŚƘţƭŚĮƴƷ ƿŤ
ƾ Śºŝ źºţ ƿ žĜ
ěǀ ŻŚ
Thrips tabaci (Thys.: Thripidae) šźĪƟřŶǀƫ
æ ƹ
*
ƽŵŚŝōźưƯƾŞŤŬƯƹ
æ ƹ
ç
æ ĭƵƹźĭ ǀ ƵŚ Īƃżě ƾ
ŻŹƹŚƄĩƵŶĪƄƳřŵ ƽ
ſƹŵźƟƵŚĮƄƳřŵ ƾ
ŶƸƄƯ
ŶƸƄƯ ç ĭƵƹźĭ ǀ ƵŚ Īƃżě ƾ
ƄĩƵŶĪƄƳřŵ ŻŹƹŚ ƽ
ŤƘƴƇƵŚĮƄƳřŵ ƾ ŵƹźƷŚƃ ŵƹźƷŚƃ
ƳƹźŤĪƫřŢƀ욌ŞţŚĪƯƩƺŘƀƯ* ǀĪ
ƾ fekrat@ferdowsi.um.ac.ir
Study on the gene expression of Chit1 and Chit2 in Beauveria bassiana during the parasitic interaction with onion thrips, Thrips tabaci (Thys.: Thripidae)
L. Fekrat1&* and M. Mammarabadi1&2
1. Department of Plant Protection, College of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran, 2. Department of Plant Protection, College of Agriculture, Shahrood University of Technology, Shahrood, Iran.
*Corresponding author, E-mail: fekrat@ferdowsi.um.ac.ir
ƵŶǀĪģ ƮƷźŝ ƱĥƺţŚěƂƴĩ
Ưǀ řźƟƱŚŝż ƿ Ŷƴ ƽ ƀŝ ǀ 쏌 ǀ Ĥ ǀ ŢſřƵŶ ƀŝŶƴƳŚưƷ ǀ ŹŚ ƽ ġŹŚƣŻř ŚƷ ƽ
šřźºƄůƱĥƺţŚºě
Beauveria bassiana
żƳō ƿƮ ŚƷ ƾƿ ƫƺţ ǀ ƯŶ ƾ ĭŵƺƫōƱŚƯŻŹŵƶĩŶƴĩ ƾ
řźŝ ƽ ƯƱŶŝƪųřŵƶŝŷƺƠƳ ǀ
ţƺĩƱŚŝż ǀ ŝŻřřŹƩƺĪ ǀ ƯƲ ƾ ŶƳźŝ ĩǀ Ťǀ żƳōŚƷŻŚƴ ƿƮ ŚƷ ƽ ưƸƯ ƾ řźŝ ƽ
Ʒǀ ƫƹŹŶ ǀ ĩż ǀŤǀ ƶŝƲ ƯŹŚưƃ ƾ ƯƹŶƳƹŹ ƾ ưƸƯƂƤƳŶƴƳřƺţ ƾ
ŝƩźŤƴĩŹŵ ǀ ĥƺƫƺ ƿ ƞƬŤŴƯšŚƟōĨ
źţƶƬưūŻř ƿ ěžĜ ǀ ŻŚ
Thrips tabaci Lind.
řƿ ŚưƳŚƠ ƿ Ŷƴ řŹŵ ƿ Ʋ ƂƷƹĦě ŚƷźĭŻŚƛō
ƽ řźŝƅŚų ƽ Ʊĥ ŚƷ ƽ CHit1 ƹ CHit2 ºůřźƏ ƾ ƺºĮƫřƹŶºƃ ƽ
ŝ ºǀ řƱŚ ºƿ źŤºſřŹŵƱĥƹŵƲ ƿ
Ʋ
IRAN 789C
ġŹŚƣ B. bassiana
ƬƛƶſŹŵ ŢƔ æåÑ
æåÒ ƹ æåÓ ƴĩ ǀŶ ƽ ƯŹŵ ǀƬ ƾ ƫǀ ŻřŹŚěƪƯŚƘţŻřŶƘŝƹƪŞƣźŤ ƿŤ
ƾ ŚƷƹŹLJŚŝ ƽ ƹŵƲſ ƭ
ŻŚǀěžĜƿźţ ƁƹŹŻřƵŵŚƠŤſřŚŝ RT-PCR
ſŹźŝ ƾ ƿŵźĭ Ŷ ƶưƷŹŵ ƽ ƮƷźŝ Ƃƴĩ
ŚƷ ŝǀ ƱĥƱŚ ŚƷ ƽ źƔƳŵŹƺƯ ŶƘŝ ŻřŹŚěƪƯŚƘţŻř ƿŤ
ƾ ŶƃƭŚŬƳř
řżƟřŚŝƹ ƿ ƴĩŢƔƬƛƂ ǀŶ
ƽ ŚƷ ƽ ƯġŹŚƣ ǀ ŝƱřż ǀ ƳƱĥƱŚ ǀż řżƟř ƿ Ƃ ƿ ŢƟŚ ŝ ǀ řƱŚ ƿ ƱĥƲ ŻřŹŚěƪƯŚƘţƭŚĮƴƷŹŵŚƷ ƿŤ
ƾ ŝŚƷƹŹLJŚŝġŹŚƣ ǀ
ŚůźĮƳŚ ƿ ż
ưƷř ǀ Ţ Ʊŵƺŝ ĩǀ Ťǀ ŚƷŻŚƴ ƶŝ ŹƺŤĩŚƟƱřƺƴƗ ƽ
Ư Œ źŧ Źŵ ŝƩźŤƴĩ ǀ ĥƺƫƺ ƿ Ĩ ƳŚĭŹř ǀ Ʈƀ ŚƷ ƽ ƯƝŶƷ ƾ ŶƃŚŝ
ƬĩƱŚĭĥřƹ ǀŶ ƽ Beauveria bassiana
ŝǀ
ƱĥƱŚ RT-PCR
Thrips tabaci
CHit1
CHit2 Abstract
The host-pathogen interaction is a very complex process. Like many entomopathogenic fungi during infection process, Beauveria bassiana produces enzymes which are able to destroy the cuticle of insects in order to penetrate into the host. Chitinases are considered to be the important enzymes for the hydrolysis of chitin and play an important role in the biological control of various pests including onion thrips, Thrips tabaci Lind. In the present study, specific primers for CHit1 and CHit2 genes were designed to study the expression pattern ofthese two chitinases from B. bassiana IRAN 789C strain before and after interaction with the second instar larvae of onion thrips using RT-PCR. Larval inoculation was carried out using three concentrations, 104, 105 and 106 conidia per ml. The results showed that both genes were expressed in all conidial concentrations, after parasitic interaction. Increasing the conidial concentration of B. bassiana caused the upregulation of chitinases expression level. Expression of these genes during the parasitic interaction of the fungus and insect larvae reflects the importance of chitinases gene as an effective factor in biological control of target organisms.
Key words: Beauveria bassiana, gene expression, RT-PCR, Thrips tabaci, CHit1, CHit2
ƶƯŶƤƯ ƩŚŝ ƦƄƿŹ ƱřŹřŵ
(Thysanoptera)
ƮƸƯƶƬưūŻř
Ʋƿźţ
ƮŤººƀǀſŹŵŢººƟōšřźººƄů
śƺººƀŰƯƽŻŹƹŚººƄƧƽŚººƷ
ƾƯ ŶƳƺƃ
(Kirk, 1997)
ŵƹŶů æåå ƲºƿřŻřƶƳƺĭ ƶŤºſřŹ
ƶŝ ƱŚƿŻšřźƄůƱřƺƴƗ
ƽŻŹƹŚƄƧšLJƺƈŰƯŹƹō ŭźºƐƯ
ŶƴŤƀƷ ƶƯŶƇŦƗŚŝƹ
ƽ ƽŶū Śƿ
ŝ ƶ ŻřŚƿƹƮǀƤŤƀƯŹƺƏ
ƢƿźƏ ƽŹŚưǀŝƩŚƤŤƳř
ƽŚºƷ ƾƷŚºǀĭ
šLJƺºƈŰƯƲºƿřƶºŝ
ƾƯ ŶƳƺƃ
(Lewis, 1997)
Ƃǀŝ ţ ź ƩŚºŝ ƦºƄƿŹ ŢºƟōƱřŹřŵ ƶŝƢƬƘŤƯ
ƵŵřƺƳŚų ƽ
Thripidae
ŶƴŤƀƷ ƶƧ ŵƹŶºůƱƺƴƧŚţ
çååå ƵŶººƃƶŤųŚƴººƃŚººǀƳŵŹŵƵŵřƺƳŚººųƲººƿřŻřƶººƳƺĭ ƹ
īŹżŝ žƴūƲƿźţ Ʊō
ƪƯŚºƃƶºƧ çìÒ
ƾºƯƶºƳƺĭ
ŶºƃŚŝ
žƴū
Thrips L.
Ţſř
(Mound, 1997)
ŚƌƗřƱŚǀƯŹŵ ƽ
ŻŚºǀěžĜƿźºţžƴūƲƿř
Thrips tabaci Lindeman
ƶºŝ
ƱŚŝżǀƯƵŵźŤƀĭŹŚǀƀŝƂƴƧřźěƪǀƫŵ
ƹŵŶƘŤƯƾƷŚǀĭƽŚƷ
ŻřƾººƨƿƽŻŹƹŚººƄƧšLJƺººƈŰƯƶººŝŶƿŶººƃšŹŚººƀų ƮƸƯ ƶŝšŚƟōƲƿźţ
ƾƯŹŚưƃ ŵƹŹ
(Ananthakrishnan, 1979;
ÓÍ šźƨƟ
ƹ ƽŵŚŝōźưƯ ŝİſŹźŝ ǀ ƱĥƱŚ įŚƷ
CHit1
ƹ
CHit2
ġŹŚƣŹŵ
Beauveria bassiana
Kirk, 1997)
ƶƳƺĭŻŚǀěžĜƿźţ ř
ƽ ƶưƷ Śū Ż ƽ ƾºƬěƹ ĥŚºƟ
ƂǀºŝƶºŝƶºƧŢſř Żř
èåå ƶºƳƺĭ Żř ºƷŚǀĭ ƱŚ ƹƾºƗřŹŻ
ƶººƳŚŴƬĭ ƵŹŚººěŚººŝƹƵŵźººĩƶººƬưůƽř
ŻřƶººƿŸƜţƹƱŵźººĩ
ƩƺƬſšŚƿƺŤŰƯ
ƶƨƫŵŚŬƿřŦƗŚŝŚƷ
ƵźºƤƳƽŚºƷ
ƽř ĬºƳŹ
ƾƯīźŝƽƹŹ
ŵƺƃ ƶƿŸƜţ ƽ ƮƷźºŝŦºƗŚŝžĜƿźţŶƿŶƃ
ƹƵŚººǀĭƾƳƺººƯŹƺƷƱŻřƺººţƱŵŹƺººų ŤƳŹŵ
ǀ ƶººŬ ƹƵŶººǀĤǀě
ƪƨƃŶŝ ƷŚºĭƹƱŶƃ ƾ
šŚºƣƹř īźºŝŶºƃŹƞºƣƺţ
ŚºƷ
Ư ƾ ƃ ŵƺ ƾºƯŻŚǀěžĜƿźţƲǀƴĤưƷ
ŽƹźºƿƹŶºƳřƺţ
ƽŚºƷ
ƽŹŚưǀŝ ƾƷŚǀĭƽřŻ
ƶºƨƫƾºĭŵźƯĦěŽƹźºƿƹƶƬưūŻř
ƽř
ƶūƺĭ ƱƺºţƺţƽŹřƺƳŽƹźƿƹƹƾĮƳźƟ
ŶºƿŚưƳƪºƤŤƴƯřŹ
(Capinera, 2001)
šLJƺƈŰƯŻřƽŹŚǀƀŝŵŹƺƯŹŵ ƩźŤƴƧ
ŻŚǀěžĜƿźţ
ľŚţŶưƗ ƵźƄůƎſƺţ
ƂƧ šŹƺƇƞƬŤŴƯƽŚƷ ƾƯ
ŵźǀĭ ƹ
LJŚŝ šŹŚƀųƱŵƺŝ
ŵŚƈŤƣř ƽ ºƃŚƳ ƾ ŦºƗŚŝŢºƟōƲºƿřŻř
ƮſƭŚŬƳř ƃŚě ƾ ŚƷ ƽ ŝƹŹźĪƯ ƾ ƱōƶºǀƬƗƶƿƹŹ ƾºƯ
ŵƺºƃ
ƵƹLjƗźƯřƲƿř
ƹƱŚºƀƳřŢƯLjºſźºŝśƺƬƐƯŚºƳšřźŧřźŝ
ƱŚƴưºƃŵƕƺºƴţƹŵřŶºƘţƂƷŚºƧŦºƗŚŝŢºƀƿŻƎǀŰƯ ƶƴƿżƷƂƿřżƟřƹŢƯƹŚƤƯŻƹźŝƾƘǀŞƏ
ƵŶƿŵźĭŶǀƫƺţƽŚƷ
Ţſř
(Coudriet et al., 1979)
ƵƹLjºƗ źºŝ Ʋƿř LJŚºŝ
Ʊŵƺºŝ
ŻŚºǀěžĜƿźºţŶƃŹŢǀƟźƓ
(Trichilo & Leigh, 1988)
ƹ
řżƟř ƿ źſƂ ƿ ŢƯƹŚƤƯƖºº Ʊō
ŢƟōƶŝ Ƃºĩ ŚºƷ ƽ ƞºƬŤŴƯ
(Van Rijn et al., 1995)
ƁƹŹźƿŚºſŻřƵŵŚƠŤºſř
ŚºƷ ƽ
ƩźŤƴĩ
ĨƿĥƺƫƺǀŝƩźŤƴĩƶƬưūŻř
řŹ ŹƹźƋ ƽ Ư ƾ ŵŻŚſ
ġŹŚƣƂƤƳŵŹƺƯŹŵ
šřźƄůƩźŤƴƧŹŵƱĥƺţŚěƽŚƷ
ººſŹźŝŢººƟō ƾ
ŢººſřƶººŤƟźĭšŹƺººƇƾººƠƬŤŴƯƽŚººƷ
(Vicentini et al., 2001; Loc & Chi, 2007; Castrillo
et al., 2008, 2010)
ŝŻř ǀ ġŹŚƣƲ ƽŚƷ źĭŹŚưǀŝ
šřźºƄů
ġŹŚƣ
Beauveria bassiana (Balsamo) Vuillemin
ƢƬƘŤƯ
ƶŤſřŹƶŝ ƽ
Hypocreales
ƶŝ ƪǀƫŵ řŹřŵ Ʊŵƺŝ ƪǀƀƳŚŤě ƪºŝŚƣ
ƽřźŝƶƔůLjƯ
ƩźŤƴƧ ŢǀƘưū
šŚƟō ƲƿźţLJŚŝ šŚƸūƺţ řŹ
ƶººŝ ƅŚººƈŤųřŵƺººų
Ƶŵřŵ Ţººſř
(Feng et al., 1994;
Castrillo et al., 2008)
Ʋººƿř ƶººƳƺĭ
Ɩǀººſƹ Ʋƿźººţ ƹ ƕƺƴŤƯ
Ʋƿźţ ƞǀƏ ƳŚºŝżǀƯ ƾ šřźºƄůŹŵřŹ řŵƞºƬŤŴƯ
ŵŹ
(Inglis et al., 2001)
ƹ ƶŝƵŻƹźƯř ƱřƺƴƗ ƿĪ ƾ ƮƸƯŻř źºţ ƿ Ʋ
ŝƩźŤƴĩƪƯřƺƗ ǀ
ĥƺƫƺ ƿ ƱŚºƸūŮƐſŹŵĨ ƵŵŚƠŤºſřŵŹƺºƯ
ƯŹřźƣ ƾ ĭǀ ŵź
(Sassa et al., 2009)
ƿ ºĪ ƾ ƵŶºưƗŻř źºţ ƿ Ʋ
ƳŚĪƯ ǀ Ʈƀ ŚƷ ƽ řƪºưƗ ºƿ ºţġŹŚºƣƲ Őŧǀ
ƹŹƱōź ƽ ŢƬĪºſř
ūŹŚų ƾ źƏŻřšřźƄů ƿ
ŮƃźţƢ żƳō ƿƮ ŚºƷ ƽ ƹŻŚºŘţƹźě
ĩǀ Ťǀ ŻŚƴ ŦƗŚŝƶƧŢſř
ţƺĩƩLjŰưºƋř ǀ
ƪºůřźƯƹƩƺºĪ
ŶƘŝ ƽ ĭŵƺƫō ƾ ƾƯ ŵƺƃ
(Sassa et al., 2009)
řŹŵ ƿ ºŝƲ ǀ Ʋ
ƳōƂƤƳ żƿ Ʈ ŚƷ ƽ żŬţ ƿƶ ƵŶºƴƴĩ ƽ ĩ ǀ ºŤ ǀ ơƺºƟƲ ƮºƸƯƵŵŚºƘƫř
ŢſřƵŶƃƵŵřŵƵƺƬū
(Fang et al., 2005)
ƱƺƴĩŚţŶƴģźƷ
ƯŚưţ ƾ Ʊĥ ŚƷ ƽ ĩ ǀŤǀ řŹŵŵƺūƺƯŻŚƴ ƿ
ŝġŹŚƣƲ ƶ ƪºƯŚĩŹƺƏ
ŚſŚƴƃ ƾƿ ƵŶƄƳ Ƴř Ŷ ƫƹ ƾ řŹŵ ƿ ƯƲ ǀ ƱŚ ŚſŚƴƃ ƾƿ ƫřƺţƹ ƾ ƿŝŚ ƾ
Ʊĥƹŵ
CHit1
ƹ
CHit2
ľLjƯŚºººƧ ŢºººſřƵŶºººƃƭŚºººŬƳř
(Fang et al., 2005)
ƯŻŹŵ ǀ ƶƴ ƽ ºţ Őŧǀ ġŹŚºƣź
B. bassiana
řźºŝ ƽ ƩźºŤƴĩ
źţ ƿ žĜ ƤŰţŚƷ ǀ ƠƬŤŴƯšŚƤ ƾ
ƳŵŹŵ ǀ šŹƺƇŚ ĭ
ŢſřƶŤƟź
ƯƶƬưūŻř ƾ ƶƘƫŚƐƯƶŝƱřƺţ
ƽ
Thungrabead et al. (2006)
ƶĩŵƺưƳƵŹŚƃř ţŐ
ƶūŹŵźǀŧ ƽ řŹƾƷŚǀĭƱŚŝżǀƯƹšŹřźů
ġŹŚººƣŻřƾººƠƬŤŴƯƕřƺººƳřƾƿřŹŚººƧƽƹŹ
ŢººƿŻřŹŚěƽŚººƷ
ƗšřźƄů ƶǀƬ
Frankliniella occidentalis (Pergande)
ƹ
T. tabaci
ŶººƳŵźƧƶººƘƫŚƐƯƾƷŚĮººƄƿŚƯŻōƎƿřźººƃŹŵ
ưƷ ǀ Ʋ ŹƺƏ
Ahmed & El-Mogy (2011)
ƱƺǀſLJƺƯźƟ
ƽŚƷ
ƞƬŤŴƯ źģ ƿ Ƃ
B. bassiana
ŚŝƺūƺūƲƛƹŹƹ
(Jojoba)
ĭŹŸŝŻřƶĩ ǀ
ƵŚ
Simmondsia chinensis (Link)
ºŝ ƶ Ţºſŵ
Ư ƾ ōƿ Ŷ ƩźŤƴƧƽřźŝřŹ
T. tabaci
ƾŝŚºƿŻŹřŻŚºǀěƕŹřżƯŹŵ
ŶƳŵźƧ ƵŹƹŵŹŵġŹŚƣƲƿřŻřƵŵŚƠŤſřƶƧŶƴŤƟźĭƶŬǀŤƳƹ
ƽ
ŝƂƤƳŚŝƺūƺūƲƛƹŹŚŝƵřźưƷŻŚǀěƵŚǀĭƾƄƿƹŹ ƶ
ƾƿřżſ
ŵŹřŵŢƟōƲƿřƩźŤƴƧƹŢǀƘưūƂƷŚƧŹŵ
řŹŵ ºƿ Ʊřź
ƹ
ƩŚſŹŵ ŚƷ ƽ ųř ǀź
ƤŰţ ǀ šŚƤ ƵŶºƴƧřźě ƽř ƹŹ ƽ ġŹŚºƣ
B.
bassiana
ººţƹ Őŧǀ ººſLJƺƯźƟź ǀƺ
Ʊ ŚººƷ ƽ ƞººƬŤŴƯ Ʊō
ƹŹ ƽ
ŚºººƷƹŹLJ ƽ ŻŚºººǀěžĜƿźºººţ ŢºººſřƵŶºººƃƭŚºººŬƳř
(Bena-Molaei et al., 2011)
ƶƬưūŻř ƾƯ ƶŝƱřƺţ ƂƷƹĦě
ƶƯŚƳ ƵźƄůƲưŬƳřƽ
ƱřźƿřƾſŚƴƃ èè
æ
ÎÐîç
ÓÎ
Ezzati-Tabrizi et al. (2009)
ºţƶƧŵƺưƳƵŹŚƃř Ő
ƕřƺºƳřźǀŧ
ƱƺǀſLJƺƯźƟ
ƞƬŤŴƯƽŚƷ
B. bassiana
ƽŚºƷƹŹLJƽƹŹřŹ
ŻŚǀěžĜƿźţ
ŶºƳŵřŵŹřźºƣƾºſŹźŝƹƶºƘƫŚƐƯŵŹƺƯ
ƢºŞƏ
ţ
ƲǀƤƤŰƯƲƿřšŚƤǀƤŰ
ƶºŝƦºưƳƹƵřźºưƷŵřƺƯƱŵƹżƟř
ƽŶǀƴƧƱƺǀƀƳŚĜſƺſ
źưƗƩƺƏƂƿřżƟřŦƗŚŝġŹŚƣƽŚƷ
ŻŚºǀěžĜƿźºţƽŚºƷƹŹLJƽƹŹƱōƽźĭŹŚºưǀŝŢǀƇŚųƹ ƾƯ ŵƺƃ ƬƗ ƾ řƮƛŹ ƿƴ ƶĪ Śţ ŻřƽŹŚǀƀŝƱƺƴĩ ƪƯřƺƗ
Źŵƪºǀųŵ
ƽŹŚưǀŝ ġŹŚƣƾƿřŻ
B. bassiana
ƶºŤƟźĭŹřźƣƶƘƫŚƐƯŵŹƺƯ
Ţſř ƴĤưƷƹ ǀ Ʋ ƁŹřżĭ ƽŚºƷ ƵŶºƴĩřźě ř ƽ ƅƺºƈųŹŵ
ƵƺŰƳ ƽ ŝ ºǀ ƱĥƱŚ ŚºƷ ƽ ĩ ǀŤǀ řŻŚºƴ ºƿ ºųźŝŹŵġŹŚºƣƲ ƾ
Żř
ƯŵƺūƺƯšřźƄů ƾ
ŶƃŚŝ ƫƹ ƾ Ʒ ǀ Ģ ſŹźŝƶƳƺĭ ƾ
ƣŵ ºǀ ƹƢ
ƏƺƀŞƯ ƾ ƵƺºŰƳƅƺƈųŹŵ
ƽ ŝ ºǀ ƱĥƱŚ ŚºƷ ƽ ųŵ ºǀ Źŵƪ
ōźƟ ƿ Ŷƴ ŝƩźŤƴĩ ǀ ĥƺƫƺ ƿĪ ƾ ƳƺĭŚŤƳōƪƯŚƘţƭŚĮƴƷ
ǀ Ťƀ ƾ ř ºƿ Ʋ
ŚŝġŹŚƣ źţ ƿ ěžĜ ǀ ŻŚ ŸĜƳšŹƺƇ ƿ
ŤƟź Ţºſřƶ Śŝţ ƶºŝƶºūƺ
ưƷř ǀ ƬĩƂƤƳƹŢ ǀŶ
ƽ żƳō ƿƮ ŚºƷ ƽ ĩ ǀŤǀ ºƇŚųŹŵŻŚºƴ ǀ
Ţ
ƳƺĭŚŤƳō ǀ Ťƀ ƾ řƿ ġŹŚƣƲ
ƬºƇřƝŶºƷ ƾ
řŹŵ ºƿ ƂƷƹĦºěƲ
ſŹźŝ ƾ ŝǀ ƱĥƱŚ ŚƷ ƽ
CHit1
ƹ
CHit2
ġŹŚºƣ
B. bassiana
ŻřŹŚěƪƯŚƘţƭŚĮƴƷ ƿŤ
ƾ ŚƷƹŹLJŚŝ ƽ ŻŚǀěžěźţ
ƵŵŚƠŤºſřŚŝ
ƁƹŹŻř
RT-PCR
Ư ƾ ŶƃŚŝ ƲǀƫƹřźƋŚůƢǀƤŰţ
ŹŵƭŚºĭ
ƀǀƳŚƨƯƥŹŵ Ʈ
ƽŹŚưǀŝŹŵƪǀųŵƾƫƺƨƫƺƯƽŚƷ
ġŹŚƣƾƿřŻ
ŹƺƧŸƯ Źŵ ŻŚǀěžĜƿźţ
ƾƯśƺƀŰƯ
ŵƺƃ
ƁƹŹƹŵřƺƯ
ŚƷ
ƽŚƷƹŹLJƁŹƹźě
ŻŚǀěžĜƿźţ
žĜƿźººţƾººƴƬƧŵŚººŬƿřƽřźººŝ
ƵŚĮººƄƿŚƯŻōŹŵŚººƷ
ƶƳƺưƳ ƶºƤƐƴƯŻŚǀěƕŹřżƯŻřƽŹřŵźŝ
ƽ ŶƸºƄƯŹŚºƈůźǀƃ
ƹŢƟźĭšŹƺƇ žĜƿźºţ
ƖºưūƽŚºƷ
ƵŶºƃƽŹƹō
ƽƹŹ
ƱřŶƬĭ ƽŚƷ Źƹźě Ɓ ŻŚǀě ŶƳŶƃƽŻŚſŚƷŹ
ƶŝ ƶºĮƳŹƺºƔƴƯ
ƮƷřźººƟƹƖºŞƴƯƾºƴƬƧƲŤºƃřŵ
ƾƟŚººƧŢºǀƘưūƱŵƺºŝ Żř
žĜƿźţ ŚƷ
ƽŵƁƹŹŻř
żƿƺƫō
(Loomans & Murai, 1997)
ŶƃƵŵŚƠŤſř ƾƴƘƿ
ƱřŶƬĭƝřźƏřŹŵ
ŚƷ ƽ ƶºŝƵŵƺºƫōŻŚºǀě žĜƿźţ
ƹŚůƱřŶƬĭƽŵřŶƘţ ƽ
ŹřźºƣƮƫŚºſŻŚºǀěƱŚºƷŚǀĭ
Ƶŵřŵ ƾƯ Ŷƃ žĜƿźţ ŚƷ ƽƺºſƶŝ ºƷŚǀĭ ŢºƧźůƮƫŚºſƱŚ
ŵźƧ Ƶ ƽƹŹƹ Ʊō ŚƷ źǀŨƨţ ƾºƯ ŶºƴŤƟŚƿ ƵŵƺºƫōƱŚºƷŚǀĭƹ
ƽ
ƶŝƾưƿŶƣ ƝŸůƹƦƄųŪƿŹŶţ ƾƯ
ŶƳŶºƃ ƹƶºƯřŵřŚºŝ
ŻŚºǀěžĜƿźºţƾŤºƀƿŻƞºƬŤŴƯƪºůřźƯŹŚƧƲƿřŹřźƨţ ŵƺŝŽźŤſŵŹŵƵŹřƺưƷ
ƽřźŝ ŚƷƹŹLJƲŤƃřŵ ƽ
ƮºƷ Ʋºſ
ƭƹŵƲſ ƶŝ
ſƹ ǀ ƶƬ ƽ ºƿ ŵřŶºƘţĨºģƺĩŹƺţřźǀĜºſōĨ
Ñå èå źţŵŶƗ ƴƬĩŻřƚƫŚŝžĜƿ ƾ
žĜƿźţ ƖưūŚƷ Źƹō ƽ ƹ
ƪųřŵ ƿ žƠƣĨ ĭźŝ ƾ ŶƳŶƃƪƤŤƴƯ
ŢƸū ſźŤſŵ ƾ ƶºŝ
ƟŚĩŵřŶƘţ ƾ řźŝŚƷƹŹLJŻř ƽ
ŹŚưǀţźƷ ŹŚƸģ
žƠƣ ĭźŝ ƾ
ŶƃƶŤƟźĭźƔƳŹŵ
žƠƣ ŚƷ ƽ ĭźŝ ƾ ŝźƯ ƶŝŻŚǀěƵŚǀĭƶŝƍƺ
ƶƳřƺŤſřƪĪƃ ŵƹŶůƩƺƏƶŝ
Ñ źƐƣƹ æ ŤƳŚſ ƾ ŻřƹźºŤƯ
ŤſLjěžƴū ǀ
ŝƵŶƃƶŤųŚſĨ ŶƳŵƺ
ƶŝ źǀĭƺºƬūŹƺºƔƴƯ ƽ
žĜƿźţŹřźƟŻř
ŚƸŤƳřƹŵŚƷ ƽ
ŵƹŶºƀƯƶŞƴěƎſƺţƶƳřƺŤſř
ƹ ƶŝ Ʊō šŶºƯƶŝƶĩŶƃƵŵřŵƵŻŚūřŚƷ çÑ
ƹŹŢƗŚºſ ƽ
īźººŝ ƮººŴţŚººƷ żººƿŹ ƽ ƴƧ Ŷººƴ ººƏŻřžººě ƾ
ƱŚººƯŻƲººƿř
žĜƿźţ ŚƷ ƽ žƠƣƪųřŵŻřƚƫŚŝ
ŚƷ ƽ ĭźŝ ƾ ŶƳŶºƃũŹŚų
ƹŚůīźŝšŚƘƐƣ ƽ
ƪºųřŵƹƵŶºƃřŶūƱŚƷŚǀĭŻřƮŴţ
Ťě ź ƽ ŚƷ ƽ ĪǀŤſLjě ƾ źƐƣƶŝ î ŤƳŚſ ƾ ƞºĩƶĩźŤƯ
Ʊō ŚºƷ
ƟŚƇŸƛŚĩƎſƺţ ƾ
ºƃƵŶƳŚƃƺěśƺƏźƯ Ŷ
ƪºƤŤƴƯŵƺºŝƵ
ŶƳŶƃ ƹŹ ƽ źŤěƲƿřŹŵ ƽ
ųřŹƺſŚƷ ƾ
ƖŝźƯ ŞƘţƪĪƃ ǀ ƹƶ
ƶģŹŚěƎſƺţ
ƽ ŶƃŵƹŶƀƯŢƟŚŝżƿŹ
řźŝ ƽ źǀĭƺƬū ƽ Żř
ƫŚưŤůřŹřźƟ ƾ
žĜƿźţ źŤěźƷŹƹŵŚƷ ƽ
ƮƬǀƟřŹŚºěŚºŝƂºƿŵ
ŶƃƶŤƀŝ ţŢƸū ŐƯ ǀ Ʋ źǀĭƺºƬūƹŢŝƺƏŹ ƽ
ĨºƄųŻř
ĭźŝšŚƘƐƣƱŶƃ ƾ
ƶŝ
ƹŹśōƶƳřŻƹŹŹƺƏ ƽ
ƟŚƇŸƛŚĩ ƾ
ƯƶƟŚƋř ƾ Ŷƃ ƵŶƷŚƄƯŢƸū
ƽ ŚºƷƹŹLJũƹźºų ƽ
Ʋºſ
ƭƹŵ ĭźŝšŚƘƐƣ ƾ
ŻƹŹźƷ Śŝ ęƺƨſƹźƨǀƯƺƿźŤſř
ŵŹƺƯ
ŶƿŵŻŚŝ Źřźƣ Ư ƾ Ɵźĭ ŶƴŤ
ƶǀƸţƹŢƄĩ
ƽ ƵŵŚƯ ƽ ƤƬţ ǀ Ű ƾ ġŹŚƣ
ƶººƿřŶūŻřƂƿŚºƯŻōƲºƿřŹŵ
ƽ
IRAN 789C
ġŹŚººƣ
B. bassiana
ƶºººǀƸţ ƯŻřƵŶºººƃ Œ
ƶºººƀſ ƽ šŚºººƤǀƤŰţ
ÓÏ šźƨƟ
ƹ ƽŵŚŝōźưƯ ŝİſŹźŝ ǀ ƱĥƱŚ įŚƷ
CHit1
ƹ
CHit2
ġŹŚƣŹŵ
Beauveria bassiana
ƵŚǀĭ Īƃżě ƾ ŶºƿŵźĭƵŵŚƠŤºſřŹƺƄĩ
ƹŹġŹŚºƣ ƽ ƎǀºŰƯ
ŢƄĩ PDA
źŤěŹŵ ƽ ŵ ºƿ Ƃ ŚºƷ ƽ ĪǀŤºſLjě ƾ ƶºŝ źºƐƣ î
ŤƳŚſ ƾ źºŤěŹŵƝřźƏřƹŢƄĩźŤƯ ƽ
ƮƬǀƟřŹŚºěƎºſƺţŚºƷ
ŶƃƶŤƀŝ źºŤěžĜºſ ƽ
ŚºƯŵŹŵŹƺţŚºŝƺĪƳřƪºųřŵŚºƷ ƽ
æ t çÒ ƶºūŹŵ ƽ ſ ŽƺǀººƀƬ ŶƳŶººƃƵŵřŵŹřźººƣ
Żřžººě
řŻŹƺĜſř ƾƿ ġŹŚƣ ŢƄĩƎǀŰƯŹŵŚƷ
ŵƹŶů è ŶºƘŝƶŤƠƷ
ŢƄĩŻř ƪƿźŤſřƎƿřźƃŹŵ
ƹ ŢºƄĩŮƐºſŵƺƷźƿŻ
Żřžººě Ɓřźººų ƪĜƫŚĪººſřŚººŝ ƶººŝ
ƶººƫƺƫ ƽ ƱƺĪƫŚººƟ Òå
ƬǀƯ ƾ źŤǀƫ ƽ ƺŤŰƯ ƽ ƹƪƿźŤſřźƐƤƯśō
Tween 80
åÒ å
ŹŶƇŹŵ ƿ ŶƃƶŤŴ žĪţŹƹŻřžě
Ʊŵźĩ
ƿŚƸƳƮŬů ƾ
ƶŝ
Òå ƬǀƯ ƾ ƳŚſŹźŤǀƫ ǀ ƵŶ ľřŵŶŬƯƹ ƪưƗ ŶºƃƭŚŬƳřžĪţŹƹ
ƶŝƱƺǀƀƳŚĜſƺſ
Ţſŵ ƵŶƯō
ƶģŹŚěŻř ƽ ƀŝ ǀ żºƿŹŹŚ ŢºƟŚŝ
ƬěžƴūŻř ƾ
źŤſř ŶºƃƵŵřŵŹƺºŞƗ
Śºţ ƷšŚºƘƐƣ ºǀ
ƹƞ
ƭƺǀƬºººƀǀƯ
ŻŚºººſřŶū ƽ
ŶƳƺºººƃ ƩƺºººŞƬĭƭLJŻř
ŹŚưºººƃ
(hemocytometer)
ƽřźººŝ ƱƺǀººƀƳŚĜſƺſŢººƔƬƛƲǀººǀƘţ
ƬƇŚů ƶ ƢǀƣŹŚŝžĜſƹŶƃƵŵŚƠŤſř
śōƎºſƺţƱŵźºĩ
řźƐƤƯ ŢƔƬƛźƿŚſƪƿźŤſ
ŚºƷ ƽ ŶºǀƴĩŻřźºƔƳŵŹƺºƯ ƽ
æåÑ
æåÒ ƹ ÎÍÓ ƽŶǀƴƧ ƯŹŵ ǀƬ ƾ ƫǀ źŤ ŶƃƶǀƸţ
ŤƀǀƳƺĭŚŤƳōƪƯŚƘţ ƾ
ƶŝ ŚƷƹŹLJŹŚưǀţŹƺƔƴƯ ƽ
ŚºŝŻŚºǀěžĜƿźºţƭƹŵƲſ
ŢƔƬƛ ŚºƷ ƽ ġŹŚºƣƞºƬŤŴƯ
B. bassiana
ƶºƫƺƫŻř
ŚºƷ ƽ
ŚƟƫ ƱƺĪ Òå ƾƬǀƯ ŶƃƵŵŚƠŤſřƽźŤǀƫ
ŢƸū ƶƿƺƸţŵŚŬƿř
ƽ
ŜſŚƴƯ ŹŵƽƹŹ ƶƫƺƫƲƿřƁƺě
ƵźƿřŵƾųřŹƺſŚƷ
ƽř ƪĪƃ
ƾŞƿźƤţźƐƣƶŝ Ĩƿ
ƾŤƳŚſ ƶǀŞƘţźŤƯ ƹ
ƽŸºƛŚĩƩŚưŤſŵŚŝ
ŵźĭŵƹŶƀƯ ƿŶ
Śţ ŚƷƹŹLJŹřźƟŻř
ƽźǀĭƺƬū ŵƺºƃ
ƪºųřŵ
ƝƹźƓ
èÒ ƾºƬǀƯ ŹŚºĭōśōźºŤǀƫ æ
ōƽƹŹƹƶºŤŴƿŹ Ʊ
ƵźƿřŵƾƟŚƇŸƛŚĩƎſƺţ
ƽř ŶºƃƵŶƳŚƃƺěƪĪƃ
žĜºſ
Ʀƿ ŵƹŶůƩƺƏƶŝŻŚǀěīźŝƶƘƐƣ Ò
æ ƾŤƳŚſ ƪųřŵźŤƯ
ŢƟźĭŹřźƣƝźƓ
ƹ æÒ ŵŶƗ ƹŹLJ ŻŚºǀěžĜƿźţƭƹŵƲſ
ƱōƽƹŹ ŶƿŵźĭƪƤŤƴƯ
Żřžºě ƫŚºĩ ǀ ƵźŞ ƵŚĮŤºſŵƱŵƺºưƳ
ƂƃŚě ŢƔƬƛ ƽŚƷ æåÑ
æåÒ ƹ æåÓ ƾƬǀƯŹŵŹƺĜſř
źºŤǀƫ ƶŝ
ƶƳŚĭřŶūŹƺƏ źƷŹŵ
ƝźƓ ºƃŚě ƵŶǀ Ŷºƃ ŢºƔƬƛźºƷ
ŹŚƸģƪƯŚƃ
ŹřźĪţ ŵƺŝ ƹ ƶſ ŹŚºưǀţ æ
śōƱƹŶºŝ
ç
ƎƤƟ ō ƹś è ƹśō
Tween 80
ƶŝżǀƳ ŶƷŚºƃƱřƺºƴƗ Źŵ
ŶƳŶƃƶŤƟźĭźƔƳ
ƶƫƺƫƂƃŚěŻřžě
ŹƺţŚŝƺĪƳřƪųřŵŚƷ
ƽŚƯŵŚŝ æ t çÒ ƶūŹŵ ƽ ŽƺǀƀƬſ
ƵŹƹŵƹ ƽ ƽŹƺºƳ æÓ
ƹƾƿŚƴƃƹŹŢƗŚſ í
ŶƳŶºƃƵŵřŵŹřźºƣƾĪƿŹŚºţŢƗŚſ
źƷŚƷƹŹLJźǀƯƹīźƯƱřżǀƯ ÏÑ
ŢƗŚºſ ƹ ƶºŝ šŶºƯ è
ƱŚƿŚěŹŵƹŶƿŵźĭŢŞŧŻƹŹ ƶºǀƬĩƭƺſŻƹŹ
ƽ ƽŚºƷƹŹLJ
ƶŝƝźƓźƷŹŵŵƺūƺƯ
ƵŹŚưƃũŹŵŚŝƹƶƳŚĭřŶūŹƺƏ
ƽ
ƶººƫƺƫƪººųřŵƶººŝŹŚººưǀţƹŹřźººĪţ
ƝŹƹŶººƴěřƽŚººƷ Ò
æ
ƾƬǀƯ ƹƪƤŤƴƯƽźŤǀƫ ƱŚƯŻŚţ
ũřźŴŤſř
RNA
Źŵ ŚŝŹżƿźƟ
ƽŚƯŵ çå ƶūŹŵ ƽ ŽƺǀƀƬſ
ƶĮƳ ŶƳŶƃƽŹřŵ
ƶƿżŬţ ƽ ƶǀƬĩ ƽ Ƶŵřŵ šŚºƠƬţƶŝƍƺŝźƯƽŚƷ ŚºƷƹŹLJ
Śŝ ƭźƳŻřƵŵŚƠŤſř
ŹřżƟř
JMP, version 8
ƶŝƹ ƱƺƯŻōšŹƺƇ
ƾƟŵŚƈţľLjƯŚĩŭźƏŜƫŚƣŹŵƪƿŹƺŤĩŚƟ ƭŚŬƳř
Ŷƃ
ũřźŴŤſř
RNA
ŢųŚſƹźĭŻŚƛōƾůřźƏ cDNA
ũřźŴŤſřƽřźŝ ƶºǀƬĩřŶŤŝřRNA
ƽ ŵƺºūƺƯƽŚºƷƹŹLJ
ƶƫƺƫźƷŹŵ
ƽ ƶŝƝŹƹŶƴěř
ƹƖƿŚºƯšŻřƎſƺţƶƳŚĭřŶūŹƺƏ
ŻřƵŵŚƠŤſřŚŝ ƬĜưſźſĨƿ
ź ƶƧ ħƺºƳ ŹŚºƧƭŚºŬƳřƽřźºŝƱō
źƔƳŵŹƺƯ ƭźƟ
Ƶŵřŵ ƵŶƃ
ŵƺŝ ŶƳŶƃŵźų
žĜſ ƵŵŚƠŤºſřŚºŝ
ĩŻř ºǀ Ţ
RNeasy Plant Mini Kit-Q74904 (Qiagen, UK)
ƹ
ƱōŹŵũŹŶººƴƯƪưƘƫřŹƺŤººſŵƢººŞƏ
ƪººĩ RNA
ũřźŴŤººſř
Ŷƿŵźĭ ŢųŚſ cDNA
Żř ƶƳƺưƳ ŚƷ ƽ RNA
ƩŚƈŰŤſř
ƵŶºƃ
ŻřƵŵŚƠŤººſřŚººŝ ĩ
ººǀ Ţ
iScript TM cDNA Synthesis Kit
(Bio-Rad, CA)
šŹƺºƇƱōŹŵũŹŶƴƯƪưƘƫřŹƺŤſŵƢŞƏƹ
Ÿě ƿ ŢƟź řźŝ ƽ ůřźƏ ƾ ƭźºƳŻřŚºƷźĭŻŚƛō
ŹřżºƟř
PRIMER3
ŵźĭƵŵŚƠŤſř ƿŶ
(Rozen & Skaletsky, 2000)
ƫřƺºţƹƭŚƳ ƾ
ŚƷźĭŻŚƛō ƽ ůřźƏ ƾ ƩƹŶūŹŵƵŶƃ æ
ŢſřƵŶƃźĩŷ
Źŵ
ŚƸƳ ƿ
Ţ ƿ Ĩ Ư ǀ ƫƹźĪ ǀ źƷŻřźŤ ƿ ŻřĨ ƶºƳƺưƳ ŚºƷ ƽ
cDNA
řźŝ ƽ ŨĪţ ǀ ŻřƵŵŚƠŤºſřŚŝź
RT-PCR
ŹřźºƣƵŵŚƠŤºſřŵŹƺºƯ
ŢƟźĭ
ƶƯŚƳ ƵźƄůƲưŬƳřƽ
ƱřźƿřƾſŚƴƃ èè
æ
ÎÐîç
ÓÐ
ƩƹŶū æ ƫřƺţ ƾ ŚƷźĭŻŚƛō ƽ ůřźƏ ƾ řźŝƵŶƃ ƽ ƱŚǀŝ Ʊĥ ŚƷ ƽ
CHit1
ƹ
CHit2
ġŹŚƣŹŵ
Beauveria bassiana
Table 1. Sequence of primers designed for the expression of Chit1 and Chit2 genes in Beauveria bassiana.
Target gene Sequence
Primer
CHit1 5'-GTCATCAATGCTGCCTTTCC-3'
BFC1 (Forward primer)
CHit1 5'-CAGTCGGTCTGAACAATGAA-3'
BRC1 (Reverse primer)
CHit2 5'-GAACGGATCTGATTCCCTCA-3'
BFC2 (Forward primer)
CHit2 5'-CTGCGGCGATAACTAAAAGC-3'
BRC2 (Reverse primer)
RT-PCR
ſŹźŝƹ ƾ ŝǀ ƱĥƱŚ ŚƷ ƽ Ťǀĩ ǀ ŻŚƴ
řźºŝ ƽ ƭŚºŬƳř
RT-PCR
ĩŻř ºǀ Ţ
AccuPower PCR
PreMix kit (Bioneer, Korea)
ƹ ƢŞƏ ũŹŶƴƯƪưƘƫřŹƺŤſŵ
ƱōŹŵ ƵŵŚƠŤſř ƃ Ŷ ſǀ ţŹřźůƪĪ ƾ
ŚºſƺƯźţƵŚĮŤſŵ ƿ
źƬĪ
ƶŝ
šŹƺƇ
°C
îÒ šŶºƯƶºŝ Ñ
ƣŵ ǀ ƶºƤ ƶŤºƃźſřƹ ŻŚºſ ƽ
ƫƹř ǀƶ
èå ºſ ǀ ºţŹřźůƪĪ ƾ
°C
îç šŶºƯƶºŝ èå
ƳŚŧ ºǀ ƶ
ƹ ƶŤƃźſř ŻŚſ ƽ
°C
Óå šŶºƯƶºŝ èå
ƳŚŧ ºǀ ƶ ƩŚºƈţř
°C
ìç šŶƯƶŝ èå ƳŚŧ ǀ ƶ ƁźŤºƀĭ
ŚƸƳŹŵƹ ºƿ Ţ
°C
ìç
šŶƯƶŝ Ò ƣŵ ǀ ƶƤ ŚºƸƳƁźŤƀĭ ƾƿ
ŵƺºŝ ƭŚºŬƳřŻřžºě
RT-PCR
ŹřŶƤƯ
Ūƴě Ư ǀ ƫƹźĪ ǀ ŚºƸƳƩƺƈŰƯŻřźŤ ƾƿ
ƹŹ ƽ
Ʃĥ ƹŶƃƵŶƳřŹŻŹŚĭō ƽŹřŵźŝźƿƺƈţ
Żř ƬºƇŚůƽŚƷŶƳŚŝ ƶ
ƵŚĮŤſŵƎſƺţ
ation gel document
ŢƟźĭšŹƺƇ
ŚŤƳ ƿ ŦŰŝƹŪ
ŚŤƳ ƿ ſŹźŝŻřƪƇŚůŪ ƾ
Ư ǀ Ʊřż ƯƹīźƯ ǀź ŚƷƹŹLJ ƽ
źţƭƹŵƲſ ƿ
ěžĜ ǀ ţŻŚ ǀ ŹŚư ŢºƔƬƛŚŝƵŶƃ
ŚºƷ ƽ ƞºƬŤŴƯ
ƴĤưƷƹġŹŚƣ ǀ
ƯƹīźƯƪĩŶƇŹŵƲ ǀ
ŹŵƵŶƃƵŶƷŚƄƯź
ţźƷ ǀ ƩƹŶūŹŵŹŚư ç
ŢſřƵŶƃƶŗřŹř
ŻřžěƩƹřŻƹŹŹŵ
ţǀ ƯŹŚư ǀ ƯƹīźƯƱřż ǀ
ƵŶƷŚƄƯź
ţŹŵƵŶƃ ǀ ŚƷŹŚư ƽ æåÒ ƹ
æåÓ ƶŝ ƴƘƯŹƺƏ ƾ Źřŵ ƽ ŝǀ ƯƹīźƯŻřƂ ǀ
ƵŶƃƵŶƷŚƄƯź
ţŹŵ ǀ ŹŚư æåÑ ƾºƬǀƯŹŵƽŶºǀƴƧ
źºŤǀƫ ŵƺºŝ ÖÒÒ ÓÕ
F =
Ð
df =
ÍÍÍÎ å
P <
ƫƹ ƾ ƵŶƷŚƄƯƝLjŤųř
ƵŶƃ ŝǀ řƲ ƿ Ʋ
ţǀ ŹŚư ţƹ ǀ ŹŚƯōƑŚŰƫŻřŶƷŚƃŹŚư ƽ
ƴƘƯ ƾ ŵƺŞƳŹřŵ ƩƹŶū
ç ţŻřžěƭƹŵŻƹŹŹŵ ǀ
ƳŹŚư ǀ ƄƯšŚƟLjŤųřż ƵŶƷŚ
ƵŶƃ
ŝǀ ţƲ ǀ ƴƘƯŚƷŹŚư ƾ ŝƹŵƺŝŹřŵ ǀ
Ƃ źţ ƿ ƯƲ ǀ ƯƹīźƯƱřż ǀ
Źŵź
ţǀ ŹŚư æåÓ ƾƬǀƯŹŵƽŶǀƴƧ
źŤǀƫ ƵŶƷŚƄƯ ƃ Ŷ ÐÒÍ ÎÒ
F =
Ð
df =
ÍÍÍÎ å
P <
ţŻřžěƭƺſŻƹŹŹŵ ǀ
ŹŚư
Ư ºǀ Ʊřż
ƯƹīźƯ ǀ ƯŚưţŹŵź ƾ ţǀ ƶŝŚƷŹŚư ƴƘƯŹƺƏ ƾ Źřŵ ƽ ŝǀ Ƃ Żřźţ
Ưǀ ƯƹīźƯƱřż ºǀ
ţŹŵź ǀ ŵƺºŝŶƷŚºƃŹŚºư
ÔÖÒ ÏÍ
F =
Ð
df =
ÍÍÍÎ å
P <
ƫƹ ƾ ƯƹīźƯ ǀ ŚƷƹŹLJź ƽ źºţ ƿ žĜ
ěǀ ţŻŚ ǀ ŹŚư ŢƔƬƛŚŝƵŶƃ
ŚƷ ƽ ŹŚƯōƑŚŰƫŻřġŹŚƣƞƬŤŴƯ ƽ
ƴƘƯƝLjŤųřƮƷŚŝ ƾ
Źřŵ ƽ ŶƴŤƃřŶƳ ƩƹŶū ç ŚƤƯŚŝ ƿ ƶƀ ƽ
ƯƹīźºƯŶºƇŹŵƪĩ ºǀ
ƵŶƷŚºƄƯź
ţŹŵƵŶºƃ ǀ
ŚºƷŹŚư ƽ
ƯƞƬŤŴƯ ƾ
ŤƳƱřƺţ ǀ ƯƝLjŤųřƶĩŢƟźĭƶŬ ǀ
ƹīźƯƱřż
Ưǀ ƵŶƷŚƄƯź
ƯŚºưţŹŵƵŶºƃ ƾ
ţǀ ŹŚºƯōƑŚºŰƫŻřŚºƷŹŚư ƽ
ƴƘƯ ƾ ţƹŵƺŝŹřŵ ǀ
ŹŚư æåÓ ƾƬǀƯŹŵƽŶǀƴƧ
źŤǀƫ ŝǀ Ƃ źºţ ƿ Ʋ
Ư ºǀ ƯƹīźºƯƱřż ºǀ
řŵřŹź ºƃ Ţ ÖÒÒ ÓÕ
F =
Ð
df =
ÍÍÍÎ å
P <
ƩƹŶū ç
ƶŝ ƬĩŹƺƏ ƾ Ư ƾ ŤƳƱřƺţ ǀ ƶŬ ƽźǀĭ ŵźƧ řżºƟřŚºŝƶĩ ƿ
Ƃ
ƴĩŢƔƬƛ ǀŶ ƽ ŚƷ ƽ ƯġŹŚƣ ǀ ƯƹīźºƯƱřż ºǀ
ŚºƷƹŹLJź ƽ
źţ ƿ ěžĜ ǀ ƳŻŚ ǀ řżƟřż ƿ ƯƂ ƾ ƿ ƴĩŢƔƬƛƹŶŝŚ ǀŶ
ƽ ƱŚƯŻƹŚƷ
źƷ ŚƷŹƺŤĩŚƟŻřƹŵ ƽ
ųŵ ǀ ƯŹŵƪ ǀ ƯƹīźƯƱřż ǀ
ŚƷƹŹLJź
ƶŝ ƯŹŚưƃ ƾ ŶƳƹŹ ƩƹŶºū ç ƤŰţŹŵ ǀ ƶŝŚºƄƯšŚºƤ
źºŧř
ŢƔƬƛ ŚƷ ƽ ţƹŚƠŤƯ ƾ ƴĩŻř ºǀ Ŷ ƽ ŚºƷ ƽ ġŹŚºƣ
B. bassiana
ŝƩźººŤƴĩŢººƸū ǀ
ĥƺƫƺ ººƿ Ĩ ƶººƴĩ ƽ ƹŵƲţŹŚººţ
ƶººƐƤƳ ř ƽ
Tetranychus urticae Koch
(Gatarayiha et al., 2010)
ƶººƳřƹźě ƽ ƶƣŚººſ Źřƺºººų
Chilo partellus (Swinhoe)
(Tefera & Pringl., 2003)
ƹ ƲǀºººƴĤưƷ ƵźºººŬƳŻ
ƽ
Nilaparvata lugens (Stål)
(Feng & Pu, 2005)
ŵŹƺºƯ
ƹŶƴŤƟźĭŹřźƣƶƘƫŚƐƯ ŶƃƆŴƄƯ
řżƟřŚŝƶĩ ƿ ŢƔƬƛƂ
ƴĩ ǀŶ ƽ ŚƷ ƽ řƿ ġŹŚƣƲ
īźƯŶƇŹŵ Ưƹ
ǀź řŹŵ ƿ Ʋ šřźƄů
Ƴǀ řżƟřż ƿ Ƃ ƿ ŢƟŚ
ÓÑ šźƨƟ
ƹ ƽŵŚŝōźưƯ ŝİſŹźŝ ǀ ƱĥƱŚ įŚƷ
CHit1
ƹ
CHit2
ġŹŚƣŹŵ
Beauveria bassiana
ƩƹŶū ç ƽŚƷƹŹLJźǀƯƹīźƯŶƇŹŵ
ŹŚưǀţŻŚǀěžĜƿźţƭƹŵƲſ
ŢƔƬƛŚŝƵŶƃ
ġŹŚºƣƞºƬŤŴƯƽŚƷ
Beauveria bassiana
ŹŚưǀţŻřžěŻƹŹƶſŚţƦƿ
Table 2. Percent mortality of the second instar larvae of onion thrips treated with different concentrations of Beauveria bassiana, one to three days after treatment.
106 Conidia per ml 105 Conidia per ml
104 Conidia per ml Control
Days after treatment
33.332 ± 4.714 b 23.332 ± 4.303 b
6.667 ± 2.720 a 3.335 ± 1.925 a
First day
44.307 ± 3.771 c 21.177 ± 4.781 b
12.28 ± 5.125 ab 5.355 ± 3.418 a
Second day
74.8 ± 11.693 b 41.665 ± 4.812 b
25.275 ± 4.101 b 1.922 ± 1.922 a
Third day
95 ± 3.19 d 65 ± 3.19 c
39.997 ± 4.714 b 10 ± 5.773 a
Total
Means in a row followed by the same letter were not significantly different (á = 0.05) (LSD test).
ƮƿżƳō ŚºƷ ƽ ĩ ǀŤǀ ƫƺƴ ǀŤ ºǀ ŚºƷŹƺŤĩŚƟŻřĨ ƽ
ųŵ ºǀ Źŵƪ
ŚƳřƺţ ƾƿ ġŹŚƣ ŚƷ ƽ ƳƺĭŚŤƳō ǀ ƶºŝŢƀ ƩźºŤƴĩƪºƯřƺƗƱřƺºƴƗ
ŝǀ ĥƺƫƺ ƿ ŝŻřŦƗŚŝƹƵŶƃśƺƀŰƯĨ ǀ
ţƺĩƲŤƟŹƲ ǀ
ƩƺĪ
Ưǀ ºƯƱŚŝż ƾ ŶƳƺºƃ
(Lorito et al., 1993; Lorito 1998)
Śºŝ
ưƷřƶŝƶūƺţ ǀ
Ţ řƿ żºƳōƲ ƿƮ ƶºƘƫŚƐƯŚºƷ
ƽ ŝ ºǀ ƱĥƱŚ ŚºƷ ƽ
Ŷĩ ƵŶƴƴĩ ƽ Ʊō ưƷřŻřŚƷ ǀ ƇŚųŢ ƾ řŸºƫŢºſřŹřŵŹƺųźŝ
ŝǀ ĩƱĥƹŵƱŚ ǀŤǀ
ŻŚƴ
CHit1
CHit2
ŻřŹŚºěƪƯŚƘţƭŚĮƴƷ ƿŤ
ƾ
ġŹŚƣ
B. bassiana
ŚƷƹŹLJƹ ƽ źºţƭƹŵƲſ ƿ
ěžĜ ºǀ ŚºŝŻŚ
ƁƹŹ
RT-PCR
ſŹźŝ ƾ ŝŮƐºſƹŶƃ ºǀ
řƱŚ ºƿ
ƱĥƹŵƲ
ŚƤƯŵŹƺƯ ƿ ŢƟźĭŹřźƣƶƀ
ƶŝ ƔƴƯ ŚƤƯŹƺ ƿ ƶƀ ƽ ŝǀ řƱŚ ƿ ƹŵƲ
řźƃŹŵƱĥ ƿ
ƭŶƗƎ ƪƯŚƘţ ġŹŚƣ ƵźƄůƹ ƹ ġŹŚºƣƪƯŚƘţ
ƵźººƄůƹ ŢººƔƬƛ
ŚººƷ ƽ æåÑ
æåÒ ƹ
æåÓ ŹŵƽŶººǀƴƧ
ƾƬǀƯ źŤǀƫ ŚƷŹƺĜſřŻř ƽ
ƸţġŹŚƣ ǀ ƶ Ŷƃ ŝƹ ǀ ƱĥƱŚ ŚƷ ƽ ơƺƟ
ƱōŹŵ ŚƯŻōƕƹźƃŹŵŚƷ ƿ
Ƃ ƵźºƄůŚºŝƪºƯŚƘţŻřƪŞƣƹ
ſŹźŝŵŹƺƯ ƾ
ŢƟźĭŹřźƣ
řŢºŰţ ºƿ řźºƃƲ ƿ ƱĥƎ ºƷ Ś ƽ
ŝźƔƳŵŹƺƯ ǀ
ŵšŹŚŞƗƶŝŶƳŶƄƳƱŚ ƿ
źĮ
ƹŹ ƽ ŻŹŚºĭōƩĥ
Ʒǀ Ģ ŶƳŚŝƶƳƺĭ ƽ ĪƄţ ǀ ŵźĮƳƪ ƿŶ ƪĪƃ A, B
Î ľLJƺưƘƯ
ġŹŚƣ ƪƸſŻřƵŹřƺưƷŚƷ
ƩƺƇƺƫř źţ ƿ ŶºƴƳŚưƷƲŝźĩƖŞƴƯƲ
ŚƷŶƴƣ ƾƿ ƔƳ ǀ řźŝżĩƺƬĭź ƽ
ţ ŐƯ ǀ ƳƲ ǀ ŚƷŻŚ ƽ ŶºƃŹ ƽ ŵƺºų
ƯƵŵŚƠŤſř ƾ ŚưƳ ƿ řźƃŹŵƹŶƴ ƿ
ƹŚƀƯƎ ƽ śŚŴŤƳřƶŝŢŞƀƳ
řƿ ƴƣƲ řŸƛƱŚŰūŹŚƷŶ ƾƿ
ºƯƱŚºƄƳ ƾ ŶºƴƷŵ ºŝ ƶ šŹŚºŞƗ
ŵƿ źĮ
řŹƺƌůŹŵ ƿ
ŚƷŶƴƣƲ ƹ ƬƗ ƾ ĩŵƺūƹƮƛŹ ǀ
ºŤ ǀ ŹŵƲ
ŰƯ ǀ Ǝ
ŤŞƛŹ ƾ ŻřƵŵŚƠŤſřƶŝ ĩǀ
Ťǀ Ʋ źƏŻřƱōƝźƈƯƹ ƿ
Ƣ
ŝǀ ƱĥƱŚ ŚƷ ƽ ĩ ǀŤǀ řŮƃźţƹŻŚƴ ƿ
żƳōƲ ƿƮ ŶºƴƷřƺŴƳƱŚƄƳŚƷ ŵřŵ
ġŹŚƣ ƯŚĮƴƷŚƷ ƾ ĩƙřźſ ǀŤǀ řźŝƲ ƽ ţ ŐƯ ǀ ŵŹƺƯƲŝźĩƲ
Ƴǀ ŻŚ ŵƺų ƴƷřƺų ƶĩŢƟŹŶ Żřźºǀƛ
ŵƖºŞƴƯƱō ƿ
źºĮ ƽ Źŵ
Ťųř ǀ ŶƴƃŚŝƶŤƃřŶƳŹŚ
(Mamarabadi et al., 2009)
řŻř ƿ Ʋ ƹŹ
řźƃŹŵ ƿ ŵŚƗƎ ƽ ƹŹġŹŚƣƶĩ ƽ
ŰƯ ǀ ŶƃŹŢƄĩƎ ƵŵźºƧ
ŵƺŝ ŚƯŻōƕƹźƃŻřƪŞƣƹ ƿ
ţƺĩŚºŝƪƯŚƘţƭŶƗƹƂ ǀ
ƩƺºĪ
ƵźƄů
ŝƭŶƗ ǀ řƱŚ ƿ ƱĥƲ ŶƃƵŶƷŚƄƯŚƷ
ƾſŹźŝŹŵ ƽŚƷ
Mamarabadi et al. (2009)
Ư ƆŴƄ
Ŷƃ ŝƶĩ ǀ ĩƱĥƱŚ ǀŤǀ ŻŚºƴ
cr-nag1
ƳƺĭŚŤƳōġŹŚºƣŹŵ ǀ
Ţºƀ
Clonostachys rosea Schroers
ººŰƯŹŵ ǀ Ǝ ŚººƷ ƽ ƹŚººů ƽ
żĩƺƬĭ
ģŚƳ ǀ ƹż ƾƫ ŰƯŹŵ ǀ Ǝ ŚƷ ƽ ƹŚů ƽ ĩ ǀ ºŤ ǀ ƹƲ ºƿ Ś Źŵ
ƎǀºººŰƯ ƵŹřƺºººƿŵƽƹŚºººůƽŚºººƷ
ġŹŚºººƣƾƫƺƬºººſƽ
Fusarium culmorum (W. G. Smith)
ŢºƟŚƿƂƿřżƟř
Źŵ
ƶºĩŶºƃƵŵřŵƱŚºƄƳƶºƘƫŚƐƯƲǀưƷ
ŻŚºƴǀŤǀĩƱĥ
cr-nag1
ƪººƯŚƘţƭŚººĮƴƷ
ġŹŚººƣ
C. rosea
ŢººƀǀƯƹƹřŚººŝ
ƽŚººƷ
Pythium ultimum Trow
ƵŹřƺºƿŵŹŵƶĩ
ŵƺºųƾºƋźƗƽ
ƲǀŤǀĩŶƣŚƟ Ţſř
ŶƿŵźĮƳƱŚǀŝ
ŶºǀƿŐţŵŹřƺƯƲƿř
ƵŶºƴƴĩ ƽ
ƶŝŪƿŚŤƳ ƱŚºǀŝƭŶƗźŝƾƴŞƯźƋŚůƂƷƹĦěŻřƵŶƯōŢſŵ
Ʊĥ ġŹŚƣŹŵŻŚƴǀŤǀĩƽŚƷ
B. bassiana
ŬƳřŻřƪŞƣ ƪƯŚƘţƭŚ
ƾƯŻŚǀěžĜƿźţƽŚƷƹŹLJŚŝƾŤƿŻřŹŚě
ŶƃŚŝ
ƶƬůźƯŹŵ ƽ
ŶƘŝ ìç ƽŚƷƹŹLJŹŚưǀţŻřžěŢƗŚſ
ŚŝŻŚǀěžĜƿźţ
ŢƔƬƛ ġŹŚºƣšƹŚºƠŤƯƽŚºƷ
B. bassiana
ƱĥƱŚǀŝ ŶºƃƾºſŹźŝŚƷ
ŵŹƺºƯŹŵ Ʊĥ
CHit1
źţLJŚºŝ ƿ Ʋ
ŝŮƐſ ǀ ţŹŵƱŚ ǀ ŹŚư æåÓ ŹƺĜºſř ƾºƬǀƯŹŵ źºŤǀƫ ƵŶƷŚºƄƯ
ŵźĭ ƿ ţƹŶ ǀ ŹŚư æåÒ ŹƺĜſř ƾºƬǀƯŹŵ źºŤǀƫ ƶºŞţŹŹŵ ƽ ƭƹŵ
ƶƯŚƳ ƵźƄůƲưŬƳřƽ
ƱřźƿřƾſŚƴƃ èè
æ
ÎÐîç
ÓÒ
ƪĪƃ æ ŝǀ ƱĥƱŚ
CHit1
(A)
Ʊĥƹ
CHit2
(B)
ŢƔƬƛƶſŹŵ æåÑ
æåÒ ƹ æåÓ ƴĩ ǀŶ ƽ ƯŹŵ ǀƬ ƾ ƫǀ źŤ
Fig. 1. Gene expression of Chit1 (A), and Chit2 (B) in three concentrations of 104, 105 and 106 conidia/ml.
ŢƃřŵŹřźƣ
Īƃ ƪ A
Î ƱřżǀƯ ŝǀ ƱĥƱŚ
CHit1
ţŹŵ ǀ ŹŚư
æåÑ ŹƺĜſř ƾƬǀƯŹŵ źŤǀƫ ƀŝ ǀ řŚŝŵƺŝƮĩŹŚ ƿ
Ʋ řŹŵƩŚů ºƿ Ʋ
ţǀ ƳŹŚư ǀ ŝźƔƳŵŹƺƯƱĥż ǀ
ŵƺºŝƵŶƃƱŚ
ƪĪºƃ A
Î Źŵ
ƱĥŵŹƺƯ
CHit2
Ƴǀ ŝż ǀ Ƃ źţ ƿ ƯƲ ǀ ŝƱřż ºǀ ţŹŵƱŚ ǀ ŹŚºư æåÓ
ŹƺĜſř ƾƬǀƯŹŵ źŤǀƫ ƵŶƷŚƄƯ ºƃ ţƹŶ ǀ ŚºƷŹŚư ƽ æåÒ ƹ æåÑ
ŹƺĜſř ƾƬǀƯŹŵ ǀƫ źºŤ ƶºŞţŹŹŵ ŚºƷ ƽ ŶºƘŝ ƽ ŶƴŤºƃřŵŹřźºƣ
ƶŝ ƽŹƺƏ źǀųřŹŚưǀţŹŵƶƧ Ưǀ
ŝƱřż ǀ ƀŝƱĥƱŚ ǀ
ŚěŹŚ ǀƿ ŵƺŝƲ
ƪĪƃ B
Î
ŚƤƯŚŝ ƿ ƶƀ ƽ Ư ǀ ŝƱřż ǀ ƱĥƱŚ ºƯŹŵŚºƷ ƾ ºƿ ŝŚǀ ŚºŝƶºĩƮ
řżƟř ƿ ġŹŚƣŢƔƬƛƂ
B. bassiana
Ư
ºǀ ŝƱřż ºǀ ƱĥƱŚ ŚºƷ ƽ
CHit1
ƹ
CHit2
Ƴǀ řżƟřż ƿ Ƃ ƿ ŢſřƶŤƟŚ ŝ
ƶ ŵšŹŚŞƗ ƿ źĮ
Ưǀ ŝƱřż ǀ ĥƱŚ Ʊ ŚºƷ ƽ ĩ ǀŤǀ řŹŵŻŚºƴ ºƿ ƳƺĭŚŤƳōġŹŚºƣƲ ǀ
Ţºƀ
ººƯ ƾ ƶººƐŝřŹŶººƳřƺţ
ƽ ƤŤººƀƯ ǀư ƾ ưĩŚººŝ ººǀ ŚƷŹƺĜººſřŢ ƽ
ƤƬţ ǀ Ů ƶºƘƫŚƐƯŚºŝƹŶƃŚŝƶŤƃřŵƵŶƃ
ƽ ŝ ºǀ řƱŚ ºƿ ƱĥƲ ŚºƷ
Ư ƾ ŵŹƹōźŝƱřƺţ ƽ
ƘưūŻř ǀ ƫƹřŢ ǀƶ ƽ řŹřġŹŚºƣ ºƿ ŵƺºưƳƶ
ŝǀ řƱŚ ƿ ƱĥƲ ŻřŹŚěƪƯŚƘţƭŚĮƴƷŹŵŚƷ ƿŤ
ƾ ŚºƷƹŹLJŚŝġŹŚƣ
ŝƭŶºƗƹ ººǀ ƱōƱŚ řźººƃŹŵŚººƷ ƿƐ
ƾ ĩƶººĩ ǀ ººŤ ǀ Ʋ ƵźººƄů Źŵ
ƳġŹŚƣŽźŤſŵ ǀ
ŝŢƀ ǀ ŚůźĮƳŚ ƿ ưƷřż ºǀ Ţ ƂºƤƳƱŵƺºŝ Ʊō
ƶººŝŚººƷ ŹƺŤĩŚººƟƱřƺººƴƗ ƽ
ººƯ Œ ŻřŹŵźŧ ººŝ ǀ Ʋ Ʊŵźººŝ
ƳŚĭŹř ǀ Ʈƀ ŚƷ ƽ ƝŶƷ ŝƩźŤƴĩƩŚưƗřƹ ǀ
ĥƺƫƺ ƿ Ĩ Ư ƾ ŶºƃŚŝ
ƾƴƘƿ ƱĥƹŵźƷ ŹƺƧŸƯ
ŝƩźŤƴĩŹŵ
ǀ ĥƺºƫƺ ƿĪ ƾ ŚºƷƹŹLJ ƽ
źţ ƿ ěžĜ ǀ ųŵŻŚ ǀ ŶƴŤƀƷƪ
ŚŤƳ ƿ ƯƶŝƍƺŝźƯŪ ǀ
ƯƹīźƯƱřż ǀ
ź ŚƷƹŹLJ žĜƿźţƽ
ŻŚǀě ŚŤƳŚŝ ƿ ŝƶºŝƍƺºŝźƯŪ ºǀ
ƱĥƱŚ ŚºƷ ƽ ĩ ǀŤǀ ƶºƐŝřŹŻŚºƴ
ƽ
ƤŤƀƯ ǀ ŵŹřŵƮ
ƾƴƘƿ řżƟřŚŝ ƿ ƴĩŢƔƬƛƂ ºǀ
Ŷ ƽ ƯŚºƷ ºǀ Ʊřż
ŝǀ ƱĥƱŚ ŚƷ ƽ
CHit1
ƹ
CHit2
řżƟř ƿ Ƃ Ư ƾ ƿ ŶŝŚ ƹ LJŚŝŦƗŚŝ
ƲŤƟŹ Ưǀ ƯƹīźƯƱřż ǀ
ŚƷƹŹLJź ƽ ƾºƯŻŚǀěžĜƿźţ
ŵƺºƃ
ŝǀ Ƃ źţ ƿ ƯƲ ǀ ŝƱřż ºǀ ƱĥƱŚ ŢºƔƬƛŹŵŚºƷ æåÓ
ƴĩ ºǀ Ŷ Źŵƽ
ƾƬǀƯ źŤǀƫ ŝƹ ǀ Ƃ źţ ƿ ƯƲ ǀ ƯƹīźƯƱřż ºǀ
ƳŚºƷƹŹLJź ºǀ
Źŵż
ưƷ ǀ ƵŶƷŚƄƯŢƔƬƛƲ ƃ
Ŷ ƾƫŚůŹŵ
ƶƧ ƮƧ źţ ƿ ƯƲ ǀ ŝƱřż ǀ ƱŚ
Ʊĥ ŚƷ ƹ żǀƳ ƯƹīźƯ ǀ ź ƶºŝƍƺºŝźƯŚƷƹŹLJ
ŢºƔƬƛ æåÑ
ƾƬǀƯŹŵƽŶǀƴƧ
źŤǀƫ ŵƺŝ
ŽŚĜſ Źřżĭ ƽ
řƿ ŚưůŚºŝƂƷƹĦºěƲ ºƿ
ƫŚºƯŢ ƾ ƭźºŤŰƯŢºƳƹŚƘƯ
ººƄƷƹĦě ƾ ººſƹŵźƟƵŚĮººƄƳřŵ ƾ
ŶƸººƄƯ ƭŚººŬƳř ººƃ ƶººĩŶ
A B
600 bp 500 bp 600bp
500bp
106 105 104 Ladder
106 105 104 Ladder
ÓÓ šźƨƟ
ƹ ƽŵŚŝōźưƯ ŝİſŹźŝ ǀ ƱĥƱŚ įŚƷ
CHit1
ƹ
CHit2
ġŹŚƣŹŵ
Beauveria bassiana
Ŷŝ ƿ Ʋ ſƹ ǀ řŻřƶƬ ƿ ŶƤţƱŚƄ ƿ ŝźĪƄţƹź ƶ
ºƯƪºưƗ ƾ ō ºƿ Ŷ Żř
Śưů ºƿ Ţ ŚººƷ ƽ ººŝ ƾ Źŵ ººƿ ƚ Śƣō ºƿ ººƬƗŶººưŰƯŽŶººƴƸƯƱŚ ƾ
ĨŞſ ų ǀż
ŽŶƴƸƯ ĨŞſƲƀŰƯ
ų ǀ ºƴĤưƷƹż ǀ
ƮƳŚºųƲ ƀŞƏƶưƏŚƟŽŶƴƸƯ
ƾ ŵřĦºƳ ƾºƏŹŵ ƭŚºŬƳř Ʋºƿř ƤŰţ ºǀ Ƣ
źĪƄţ ƹ ƳřŵŹŶƣ ƾ Ư ƾ ƺƃ ŵ
ƖŝŚƴƯ
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ƶƯŚƳ ƵźƄůƲưŬƳřƽ
ƱřźƿřƾſŚƴƃ èè
æ
ÎÐîç
ÓÔ
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