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Activity of Azoxystrobin and SHAM to Four Phytopathogens
Agricultural Sciences in China
July 2009
2009, 8(7): 835-842
Activity of Azoxystrobin and SHAM to Four Phytopathogens JIN Li-hua*, CHEN Yu*, CHEN Chang-jun, WANG Jian-xin and ZHOU Ming-guo College of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, P.R.China
Abstract The study was conducted to make clear the activity of azoxystrobin to 4 plant pathogens and the synergistic effects of salicylhydroxamic acid (SHAM), which acted on the alternative oxidase. It was also conducted to be aware of the mechanism of azoxystrobin in inhibition on mycelial respiration and the influence of SHAM. The activity test of azoxystrobin and SHAM was carried out with a mycelial linear growth test and spore germination test. Other related biological properties were also observed. Inhibition of azoxystrobin and SHAM on 4 pathogens was determined by using SP-II oxygraph system. Azoxystrobin inhibited mycelial growth in Colletotrichum capsici, Botrytis cinerea, Rhizoctonia solani, and Magnaporthe grisea, respectively; it also inhibited conidia germination, and conidia production in C. capsici, B. cinerea M. grisea, and sclerotia formation in R. solani. Moreover, it created stayed pigment biosynthesis in C. capsici and M. grisea somehow. Salicylhydroxamic acid enhanced inhibition by azoxystrobin. An oxygen consuming test of the mycelia showed that azoxystrobin inhibited all the 4 fungi’s respiration in the early stages. With the concentration rising up, the effectiveness increased. However, as time went on, the respiration of the mycelia treated with fungicides recovered and SHAM could not inhibit the oxygen consuming. This reaction between the mycelia and the fungicides appeared not to initiate alternative respiration but rather the other mechanism created a lack of efficacy. Key words: azoxstrobin, salicylhydroxamic acid (SHAM), plant pathogens, spore germination, mycelial growth, oxygen consumption rate
INTRODUCTION Strobilurin fungicides including azoxystrobin inhibited mitochondrial electron transferring by binding to the Qo center of cytochrome bc1 complex and interfered with ATP synthesis (Ulrich et al. 1988; Ypema and Gold 1999; Xiao et al. 2003; Esser et al. 2004). The mechanism of this class of fungicides was new and they had no cross-resistance with demethylation inhibitors (DMIs), dicarboximide fungicides, and benzimidazoles, and these fungicides were used worldwide. Azoxystrobin was first registered for controlling pepper anthracnose, cucumber downy mildew, and tomato early blight in China. In other countries, it was also
used for controlling rice blast, rice sheath blight, and grey mold of cucurbit crops. However, several plant pathogens could express resistance to these fungicides by the induction of alternative oxidase pathway, which indicated that mitochondrial electron transfer with the alternative oxidase pathway activity and thus, ATP synthesis is sustained by circumvention of the cytochrome b site of QoI action (Vanlerberghe and Mcintosh 1997; Joseph-Horne and Hollomon 2000; Joseph-Horne et al. 2001). This resistance mechanism appeared in plant pathogens such as Magnaporthe grisea, Septoria tritici, Botrytis cinerea, and Venturia inaequalis (Kim et al. 2003; Ziogas et al. 1999; Tamura et al. 1999; Zheng et al. 2000). But alternative oxidase pathway, which played an impor-
This paper is translated from its Chinese version in Scientia Agricultura Sinica. Correspondence ZHOU Ming-guo, Professor, Tel: +86-25-84395641, E-mail: [email protected] *
These authors contributed equally to this study. © 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60285-0
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tant role in vitro, did not decrease the efficacy in the field application of QoIs (Kim et al. 2003; Ziogas et al. 1999; Tamura et al. 1999; Zheng et al. 2000). Olaya and Mizutani assumed that plant antioxidants such as flavone presented in the host quenched the active oxygen species from reaching the level sufficient for inducing alternative oxidase pathway during QoI action (Olaya et al. 1998; Olaya and Köller 1999; Mizutani et al. 1996). This essay explored the activity of azoxystrobin with or without salicylhydroxamic acid (SHAM), which acted as an inhibitor of alternative oxydase to 4 plant pathogens as Colletotrichum capsici, Rhizoctonia solani, Magnaporthe grisea, and Botrytis cinerea in order to explore the synergistic effect of SHAM. The objectives of this study were to: (i) determine the rate of mycelial respiration of these 4 plant pathogenic fungi when treated with azoxystrobin alone or mixed with SHAM; (ii) make clear of the exact time when the alternative oxidase pathway was started; (iii) investigate the relationship between the startup of alternative oxidase pathway and the sensitivity to azoxystrobin.
MATERIALS AND METHODS Pathogens C. capsici, R. solani, M. grisea, and B. cinerea were all isolated from wild-type single-spore isolates.
Fungicides Azoxystrobin in technical grade (93%) was provided by Syngenta, China, and dissolved in methanol to 10 mg mL-1 for stock solution. Salicylhydroxamic acid (SHAM) in technical grade (99%) was purchased from Acros Organics, USA, and dissolved in methanol to 50 mg mL-1 for stock solution.
JIN Li-hua et al.
AEA medium: 20 g of agar was added to 1 L AEB. PSA medium: 200 g of potato, 20.0 g of sucrose, 20.0 g of agar, and deionized H2O added to 1 L. WA medium: 20 g of agar was added to 1 L deionized H2O.
Sensitivity of these 4 plant pathogens to azoxystrobin Test of inhibition on mycelial growth Fresh mycelial plugs from colony margins of these 4 fungi were transferred to AEA plates amended with 0, 0.1, 0.5, 1, 5, 10, 50, 100 g mL-1 azoxystrobin alone or mixed with 150 g mL-1 SHAM at 25°C. The EC50 values for inhibiting mycelial growth were calculated according to the regress equation (Y = a + bX). Test of inhibition on conidial germination Conidia of these 4 fungi were collected and adjusted to 1.0 × 105 conidia mL-1 with sterile deionized H2O. 50 microliters of the conidial suspension was spread to the WA plates amended with 0.00625, 0.0125, 0.025, 0.05, 0.1, and 0.2 g mL-1 azoxystrobin alone or mixed with 100 g mL-1 SHAM at 25°C (100 g mL-1 SHAM could inhibit the alternative oxidase pathway completely). The ratio of the conidia germination was observed after 10 h under a microscope (20 × 10). The EC50 values for inhibiting conidia germination were calculated according to the regress equation (Y = a + bX).
Morphological characters of colony type, conidia type, and pigment biosynthesis of these 4 fungi treated with azoxystrobin and SHAM The 4 fungi were treated as described above. Morphological characters including colony type, conidia type, and pigment biosynthesis were observed after 3-15 d.
Test of sporulation and sclerotia formation capacity
Media AEB medium: 5.0 g of yeast powder, 6.0 g of NaNO3, 1.5 g of KH2PO 4, 0.5 g of KCl, 0.25 g of MgSO4, 20 mL of glycerol (99.9%), and deionized H2O added to 1 L.
Fresh mycelial plugs from colony margins of C. capsici were transferred to AEA plates with a series of concentrations of azoxystrobin alone or mixed with 150 g mL-1 SHAM. The conidia from each plates were flushed into 10 mL deionized H2O before they were calcu-
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Activity of Azoxystrobin and SHAM to Four Phytopathogens
lated with hemacytometer, respectively, after 10 and 20 d. The sporulation capacity of B. cinerea was tested as that of C. capsici after 7 d. R. solani was treated the same as C. capsici and B. cinerea. The production and fresh weight of sclerotia were measured after 20 d.
Impact of azoxystrobin on mycelial respiration of these 4 fungi Test of impact of azoxystrobin at different concentrations on mycelial respiration of these 4 fungi cultured for different time Ten mycelial plugs of these 4 fungi from colony margins were transferred to each conical flask containing 100 mL AEB for shake culture (25°C, 120 r/min). The mycelia of the 4 fungi were treated with azoxystrobin at the ultimate concentration of 0, 1, 10, and 50 g mL-1 for 1 h after 1, 3, and 5 d and then the rate of the mycelial oxygen consumption was tested with oxygraph system and the inhibition of respiration was calculated. Test of inhibition of respiration on mycelia by azoxystrobin at different treatment time After cultured for 7 d, 10 mycelial plugs of these 4 fungi from colony margins were transferred to each of the 6 conical flasks containing 100 mL AEB for shake culture, respectively (25°C, 120 r/min). Azoxystrobin was added to 3 of the 6 conical flasks with the ultimate concentration of 10 g mL-1 36 h later. Two mycelial plugs were taken out from each conical flask after shake culture for 1, 6, 12, 24, 48, and 96 h, one was treated with 150 g mL-1 SHAM and the other one was treated with deionized H2O for 1 h. And then, the ratio of the mycelial oxygen consumption was tested by oxygraph system and the inhibition of respiration on mycelia was calculated.
RESULTS Sensitivity of mycelia and conidia of the 4 fungi to azoxystrobin Test of inhibition on mycelial growth Sensitivity of the 4 fungi to azoxystrobin was determined in vitro. With the concentration of azoxystrobin rising up, the
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inhibition got enhanced and the dose-reaction curve was prominently related. Azoxystrobin had an apparent inhibition on mycelial growth of R. solani and M. grisea with the respective EC50 value 0.008 and 0.013 g mL -1. Inhibitions of azoxystrobin on mycelial growth of C. capsici and B. cinerea were weaker with the respective EC50 values of 10.907 and 70.974 g mL-1. Salicylhydroxamic acid enhanced inhibition of azoxystrobin on mycelial growth. With the application of both azoxystrobin and SHAM, these 4 fungi became more sensitive, especially for C. capsici and R. solani. In the presence of 100 g mL-1 SHAM, the EC50 values of azoxystrobin on R. solani, M. grisea, C. capsici, and B. cinerea were 0.006, 0.007, 0.0268, and 0.329 g mL-1 with the synergistic ratios 1.33, 1.86, 40.40, and 215.73, respectively. Inhibition on conidial germination Azoxystrobin inhibited conidia germination of C. capsici, B. cinerea, and M. grisea with the EC50 values 32.3147, 3.6855, and 0.094 g mL -1. However, the EC 50 values of conidial germination were 0.068, 0.103, and 0.004 g mL-1 in the presence of 100 g mL-1 SHAM with the synergistic ratios 469.24, 35.79, and 23.5, respectively (Table).
Influence of azoxystrobin alone or mixed with SHAM on colony type, conidia type, and pigment biosynthesis Azoxystrobin used alone or mixed with SHAM had different influence on colony type, conidia type, and pigment biosynthesis of the 4 fungi (Fig.1). Once C. capsici was treated with azoxystrobin, the hyphae became sparser and the xanthein was biosynthesized, which could be accelerated by SHAM. Salicylhydroxamic acid alone could not affect pigment biosynthesis. Azoxystrobin inhibited the length and width of the germling tube but did not cause malformation. Azoxystrobin used alone or mixed with SHAM could reduce the airy hyphae of B. cinerea. When B. cinerea was treated with 50 g mL-1 azoxystrobin alone, the airy hyphae were very few. When cooperated with 150 g mL-1 SHAM, only 5 g mL-1 azoxystrobin could inhibit airy hyphae prominently. Azoxystrobin alone could not affect the pigment bio-
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Table EC50 of azoxystrobin alone or with SHAM against mycelial growth and conidial germination of four pathogens Stage
Pathogens
Mycelial growth
Conidial germination
EC50 (g mL-1) - SHAM
Colletotrichum capsici Rhizoctonia solani Magnaporthe grisea Botrytis cinerea Colletotrichum capsici Magnaporthe grisea Botrytis cinerea
EC50 (g mL-1) + SHAM
Factor
0.268 0.006 0.007 0.329 0.068 0.004 0.103
40.40 1.33 1.86 215.73 469.24 23.5 35.79
10.907 0.008 0.013 70.974 31.908 0.094 3.686
(+) and (-) mean present and absent 100 (g mL-1) SHAM, respectively.
A
B CK
0.1 g mL-1
1 g mL-1
CK
0.1 g mL-1 1 g mL-1
10 g mL -1 50 g mL-1 100 g mL-1 10 g mL-1 50 g mL-1 100 g mL-1 CK
0.00625 g mL
-1
C
D 0.00625 g mL-1 0.0125 g mL-1
CK
CK
Azo
SHAM 0.025 g mL-1
0.05 g mL-1
0.1 g mL-1
Fig. 1 Influence of azoxystrobin on the tube characteristics, colony properties, and pigment biosynthesis of the plant pathogens. A, influence of azoxystrobin on germ tub growth of Collethotrichum capsici; B, influence of azoxystrobin used alone (first line) or mixed with SHAM (second line) on the colony properties and pigment biosynthesis of Collethotrichum capsici; C, influence of azoxystrobin and SHAM on the colony properties and pigment biosynthesis of Botrytis cinerea; D, influence of azoxystrobin on the clone properties and pigment biosynthesis of Magnaporthe grisea.
synthesis of B. cinerea but could make the isolate become light brown when cooperated with SHAM. Azoxystrobin used alone or mixed with SHAM could delay the biosynthesis of pigment in M. grisea but hardly affect colony type of R. solani.
Influence on sporulation and sclerotia forming Sporulation of C. capsici was very sensitive to azoxystrobin. The 0.1 g mL-1 azoxystrobin used alone or mixed with 150 g mL-1 SHAM could completely inhibit acervuli from forming. There was no conidiophore production when C. capsici was cultured on plates amended with fungicide even at each concentration after 10 d. Twenty days later, very few (8.7 × 105 conidia
mL -1) conidiophore were produced only in plates amended with 0.1 g mL-1 azoxystrobin alone, which was 99.98% less than the control whose production was 4 × 109 conidia mL-1. In B. cinerea, the production of conidiophore decreased with the concentration of fungicide rising up. The dose-reaction curve was prominently related. When azoxystrobin acted alone, the EC50 value of inhibiting sporulation was 1.1246 g mL-1; while cooperated with SHAM, the EC50 value was 0.0409 g mL-1 at the presence of SHAM. Sclerotia production of R. solani also decreased as the concentration of azoxystrobin rose up. The dosereaction curve was prominently related. The EC50 value of inhibiting sclerotia forming was only 0.0079 g
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Activity of Azoxystrobin and SHAM to Four Phytopathogens
mL-1. When the concentration of azoxystrobin was above 0.5 g mL-1, there was no sclerotia production, either.
Impact of azoxystrobin on mycelial respiration of these 4 fungi Effect of azoxystrobin on mycelial respiration of the 4 fungi of different stages The inhibition effectiveness of azoxystrobin on C. capsici was the best in all of the 4 fungi after 1 h with the inhibition ratio all above 60% in mycelia of different culture time (Fig.2). In contrast, the maximum inhibition was less than 60% for B. cinerea. The inhibition of azoxystrobin on the respiration of mycelia got stronger as the concentration rose up in all 4 fungi. In a word, azoxystrobin had an inhibition on the mycelial respiration of the 4 fungi more or less when they were treated with azoxystrobin for 1 h. Effect of respiration inhibition on mycelia of the 4 fungi by azoxystrobin at different treatment time Azoxystobin alone or mixed with SHAM exhibited different activity against the mycelial respiration of these 4 fungi (Fig.3). The mycelial respira-
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tion of C. capsici and M. grisea were more sensitive to azoxystrobin. As time went on, sensitivity of mycelial respiration of C. capsici and M. grisea to azoxystrobin decreased and was completely lost after 48 and 24 h, respectively. The mycelial respiration of R. solani and B. cinerea were less sensitive to azoxystrobin, and the former showed unchanged sensitivity of mycelial respiration to azoxystrobin, whereas the latter showed similar sensitivity as M. grisea and completely lost the sensitivity of mycelial respiration to azoxystrobin after 12 h. The data on treatment of the inhibitor of alternative oxydase SHAM and its synergistic effects with azoxystrobin indicated that as there were inherent alternative oxidase pathways in R. solani, B. cinerea, and C. capsici, single use of SHAM could inhibit the mycelial respiration of these 3 fungi. But there was no inherent alternative oxidase pathway in M. grisea, single use of SHAM showed no inhibitory activity against mycelial respiration of this fungus. However, alternative oxidase pathway appeared after 12 h treatment with azoxystrobin and strengthened as the time went on, indicating that alternative oxidase pathway could be induced by azoxystrobin in M. grisea.
Fig. 2 Inhibition of oxygen consumption in 4 fungal mycelia by azoxystrobin. A, inhibition of oxygen consumption in Colletotrichun capsici in different aged mycelia by azoxystrobin at different concentrations; B, inhibition of oxygen consumption in Rhizoctonia solani in different aged mycelia by azoxystrobin at different concentrations; C, inhibition of oxygen consumption in Magnaporthe grisea in different aged mycelia by azoxystrobin at different concentrations; D, inhibition of oxygen consumption in Botrytis cinerea in different aged mycelia by azoxystrobin at different concentrations.
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Fig. 3 Inhibition of oxygen consumption in 4 fungal mycelia treated with azoxystrobin or azoxstrobin plus SHAM at different time. A, inhibition of oxygen consumption in Colletotrichun capsici; B, inhibition of oxygen consumption in Rhizoctonia solani; C, inhibition of oxygen consumption in Magnaporthe grisea; D, inhibition of oxygen consumption in Botrytis cinerea.
DISCUSSION Since Zeneca Company (UK) developed the first commercial fungicide of strobilurins in 1996, the researches in this class of fungicides were extensive. It was reported that the spores of C. capsici were more sensitive to azoxystrobin than that in mycelia; and moreover, SHAM exhibited synergistic effects on inhibiting conidial germination but did not exhibit synergistic effects in inhibiting mycelial growth (Li et al. 2005). This study also showed that the spores of C. capsici were more sensitive to azoxystrobin than that in mycelia; however, SHAM exhibited synergistic effects on inhibiting both conidial germination and mycelial growth. Sierotaki et al. (2000) also found that trifloxystrobin mixed with SHAM had a better inhibition on the mycelial growth of Mycosphaerella fijiensis than trifloxystrobin alone. Karadimosa et al. (2005) also found that Cercospora beticola were more sensitive to trifloxystrobin than its mycelia. Mizutani et al. (1995, 1996) found that when M. grisea was treated with metominostrobin, the content of active oxygen species increased and the activity of alternative oxidase got stronger, and SHAM enhanced the effectiveness of azoxystrobin by preventing the alternative oxidase pathway from induced. Previous studies showed that on the plates of me-
dium for M. grisea to produce spores amended with 0.01 g mL-1 azoxystrobin, the spore production was 1.57 × 106 spores per plate, decreasing by 43.7% when compared with the nonamended control and the pigment biosynthesis was inhibited (Zhang et al. 2005). In this study, although spore germination of M. grisea was strongly inhibited by azoxystrobin (similar to the previous results), the pigment biosynthesis of M. grisea was only delayed by azoxystrobin. Besides inhibiting the mycelial growth and conidial germination, azoxystrobin with/without SHAM could also affect the conidia and sclerotia production of these pathogens. So it could play an important role during the whole life cycles of many pathogens and could resist pathogens from infecting and expanding if the hosts had already been infected. It could also reduce the offspring and the reinfection source so as to control the diseases effectively. Therefore, when azoxystrobin was protectively used in the field, plant antioxidants present in the host quenched the active oxygen species from reaching the level sufficient for inducing alternative oxidase pathway so as to control the plant diseases effectively. And it was much better to add SHAM according to the field conditions during the in vitro test. Also, it was suggested to protectively use azoxystrobin so as to control the plant diseases effectively based on our results. Moreover, Avila-Adame and Köller (2003)
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Activity of Azoxystrobin and SHAM to Four Phytopathogens
found that protective use of azoxystrobin could not only better control the plant disease (than curative use of azoxystrobin) but also prevent the alternative oxidase pathway during conidia germination so as to delay the development of fungicide resistance. Oxygen consumption test of mycelia in this essay showed that azoxystrobin inhibited mycelial respiration of these 4 pathogens to different extent in the early stages and the inhibition on mycelia of C. capsici was the strongest. And the inhibition effectiveness became better with the concentration of azoxystrobin rising up. However, as time went on, the respiration of the mycelia treated with azoxystrobin got stronger and SHAM could not reduce the oxygen consumption. The authors speculated that the strengthened expression of cyt bc1 complex genes might make the fungi insensitive to the fungicide. As reported by Bourges et al. (2005), the QoI inhibitor myxothiazol could change the expression of many genes, such as cytoplast catalase gene, the transformation factor Yap1, and so on. Whether this was caused by the strengthened expression of cyt bc1 complex genes should be further explored. And moreover, recently, Chen et al. (2009) found that the enhanced cyt b gene expression might play a role in the development of resistance to azoxystrobin in C. capsici. Results above also indicated that mycelia respiration had indirect relationship with mycelia growth. Mizutani et al. (1995) also reported that asexual growth hardly depended on respiration. Thus, the authors also speculated that azoxystrobin might affect other metabolisms except energy system, which is also needed to be further studied.
CONCLUSION This study presented the effect of single use of azoxystrobin or with SHAM on the sensitivity, biological properties, sporulation, or sclerotia forming, and mycelial respiration of the 4 fungi. Some conclusions could be drawn as belows: (1) Azoxystrobin could inhibit the mycelial growth of all of the 4 fungi, and the conidial germination of three of the 4 fungi. And SHAM exhibited synergistic effects with azoxystrobin. (2) Azoxystrobin affected the colony type and conidia production of C. capsici and B. cinerea, sclerotia pro-
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duction of R. solani, prevented the pigment biosynthesis of C. capsici, and delayed pigment biosynthesis of M. grisea. (3) Single use of azoxystrobin could inhibit the mycelial respiration of all the 4 fungi. The inhibition increased as the concentration increased. And as the time went on, the inhibition of azoxystrobin decreased and disappeared in these fungi except R. solani. (4) SHAM showed little synergistic effects against mycelial respiration of these fungi. The expression of alternative oxidase pathway was induced by 12 h treatment of azoxystrobin in M. grisea; however, alternative oxidase pathway was constitutively expressed in other 3 fungi.
Acknowledgements This study was sponsored by the National 973 Program of China (2009CB118906, 2006CB101907), the National 863 Program of China (2006AA10A211, 2008AA10Z414), and the National Natural Science Foundation of China (30671048, 30671384).
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2009, 8(7): 835-842
Activity of Azoxystrobin and SHAM to Four Phytopathogens JIN Li-hua*, CHEN Yu*, CHEN Chang-jun, WANG Jian-xin and ZHOU Ming-guo College of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, P.R.China
Abstract The study was conducted to make clear the activity of azoxystrobin to 4 plant pathogens and the synergistic effects of salicylhydroxamic acid (SHAM), which acted on the alternative oxidase. It was also conducted to be aware of the mechanism of azoxystrobin in inhibition on mycelial respiration and the influence of SHAM. The activity test of azoxystrobin and SHAM was carried out with a mycelial linear growth test and spore germination test. Other related biological properties were also observed. Inhibition of azoxystrobin and SHAM on 4 pathogens was determined by using SP-II oxygraph system. Azoxystrobin inhibited mycelial growth in Colletotrichum capsici, Botrytis cinerea, Rhizoctonia solani, and Magnaporthe grisea, respectively; it also inhibited conidia germination, and conidia production in C. capsici, B. cinerea M. grisea, and sclerotia formation in R. solani. Moreover, it created stayed pigment biosynthesis in C. capsici and M. grisea somehow. Salicylhydroxamic acid enhanced inhibition by azoxystrobin. An oxygen consuming test of the mycelia showed that azoxystrobin inhibited all the 4 fungi’s respiration in the early stages. With the concentration rising up, the effectiveness increased. However, as time went on, the respiration of the mycelia treated with fungicides recovered and SHAM could not inhibit the oxygen consuming. This reaction between the mycelia and the fungicides appeared not to initiate alternative respiration but rather the other mechanism created a lack of efficacy. Key words: azoxstrobin, salicylhydroxamic acid (SHAM), plant pathogens, spore germination, mycelial growth, oxygen consumption rate
INTRODUCTION Strobilurin fungicides including azoxystrobin inhibited mitochondrial electron transferring by binding to the Qo center of cytochrome bc1 complex and interfered with ATP synthesis (Ulrich et al. 1988; Ypema and Gold 1999; Xiao et al. 2003; Esser et al. 2004). The mechanism of this class of fungicides was new and they had no cross-resistance with demethylation inhibitors (DMIs), dicarboximide fungicides, and benzimidazoles, and these fungicides were used worldwide. Azoxystrobin was first registered for controlling pepper anthracnose, cucumber downy mildew, and tomato early blight in China. In other countries, it was also
used for controlling rice blast, rice sheath blight, and grey mold of cucurbit crops. However, several plant pathogens could express resistance to these fungicides by the induction of alternative oxidase pathway, which indicated that mitochondrial electron transfer with the alternative oxidase pathway activity and thus, ATP synthesis is sustained by circumvention of the cytochrome b site of QoI action (Vanlerberghe and Mcintosh 1997; Joseph-Horne and Hollomon 2000; Joseph-Horne et al. 2001). This resistance mechanism appeared in plant pathogens such as Magnaporthe grisea, Septoria tritici, Botrytis cinerea, and Venturia inaequalis (Kim et al. 2003; Ziogas et al. 1999; Tamura et al. 1999; Zheng et al. 2000). But alternative oxidase pathway, which played an impor-
This paper is translated from its Chinese version in Scientia Agricultura Sinica. Correspondence ZHOU Ming-guo, Professor, Tel: +86-25-84395641, E-mail: [email protected] *
These authors contributed equally to this study. © 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60285-0
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tant role in vitro, did not decrease the efficacy in the field application of QoIs (Kim et al. 2003; Ziogas et al. 1999; Tamura et al. 1999; Zheng et al. 2000). Olaya and Mizutani assumed that plant antioxidants such as flavone presented in the host quenched the active oxygen species from reaching the level sufficient for inducing alternative oxidase pathway during QoI action (Olaya et al. 1998; Olaya and Köller 1999; Mizutani et al. 1996). This essay explored the activity of azoxystrobin with or without salicylhydroxamic acid (SHAM), which acted as an inhibitor of alternative oxydase to 4 plant pathogens as Colletotrichum capsici, Rhizoctonia solani, Magnaporthe grisea, and Botrytis cinerea in order to explore the synergistic effect of SHAM. The objectives of this study were to: (i) determine the rate of mycelial respiration of these 4 plant pathogenic fungi when treated with azoxystrobin alone or mixed with SHAM; (ii) make clear of the exact time when the alternative oxidase pathway was started; (iii) investigate the relationship between the startup of alternative oxidase pathway and the sensitivity to azoxystrobin.
MATERIALS AND METHODS Pathogens C. capsici, R. solani, M. grisea, and B. cinerea were all isolated from wild-type single-spore isolates.
Fungicides Azoxystrobin in technical grade (93%) was provided by Syngenta, China, and dissolved in methanol to 10 mg mL-1 for stock solution. Salicylhydroxamic acid (SHAM) in technical grade (99%) was purchased from Acros Organics, USA, and dissolved in methanol to 50 mg mL-1 for stock solution.
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AEA medium: 20 g of agar was added to 1 L AEB. PSA medium: 200 g of potato, 20.0 g of sucrose, 20.0 g of agar, and deionized H2O added to 1 L. WA medium: 20 g of agar was added to 1 L deionized H2O.
Sensitivity of these 4 plant pathogens to azoxystrobin Test of inhibition on mycelial growth Fresh mycelial plugs from colony margins of these 4 fungi were transferred to AEA plates amended with 0, 0.1, 0.5, 1, 5, 10, 50, 100 g mL-1 azoxystrobin alone or mixed with 150 g mL-1 SHAM at 25°C. The EC50 values for inhibiting mycelial growth were calculated according to the regress equation (Y = a + bX). Test of inhibition on conidial germination Conidia of these 4 fungi were collected and adjusted to 1.0 × 105 conidia mL-1 with sterile deionized H2O. 50 microliters of the conidial suspension was spread to the WA plates amended with 0.00625, 0.0125, 0.025, 0.05, 0.1, and 0.2 g mL-1 azoxystrobin alone or mixed with 100 g mL-1 SHAM at 25°C (100 g mL-1 SHAM could inhibit the alternative oxidase pathway completely). The ratio of the conidia germination was observed after 10 h under a microscope (20 × 10). The EC50 values for inhibiting conidia germination were calculated according to the regress equation (Y = a + bX).
Morphological characters of colony type, conidia type, and pigment biosynthesis of these 4 fungi treated with azoxystrobin and SHAM The 4 fungi were treated as described above. Morphological characters including colony type, conidia type, and pigment biosynthesis were observed after 3-15 d.
Test of sporulation and sclerotia formation capacity
Media AEB medium: 5.0 g of yeast powder, 6.0 g of NaNO3, 1.5 g of KH2PO 4, 0.5 g of KCl, 0.25 g of MgSO4, 20 mL of glycerol (99.9%), and deionized H2O added to 1 L.
Fresh mycelial plugs from colony margins of C. capsici were transferred to AEA plates with a series of concentrations of azoxystrobin alone or mixed with 150 g mL-1 SHAM. The conidia from each plates were flushed into 10 mL deionized H2O before they were calcu-
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Activity of Azoxystrobin and SHAM to Four Phytopathogens
lated with hemacytometer, respectively, after 10 and 20 d. The sporulation capacity of B. cinerea was tested as that of C. capsici after 7 d. R. solani was treated the same as C. capsici and B. cinerea. The production and fresh weight of sclerotia were measured after 20 d.
Impact of azoxystrobin on mycelial respiration of these 4 fungi Test of impact of azoxystrobin at different concentrations on mycelial respiration of these 4 fungi cultured for different time Ten mycelial plugs of these 4 fungi from colony margins were transferred to each conical flask containing 100 mL AEB for shake culture (25°C, 120 r/min). The mycelia of the 4 fungi were treated with azoxystrobin at the ultimate concentration of 0, 1, 10, and 50 g mL-1 for 1 h after 1, 3, and 5 d and then the rate of the mycelial oxygen consumption was tested with oxygraph system and the inhibition of respiration was calculated. Test of inhibition of respiration on mycelia by azoxystrobin at different treatment time After cultured for 7 d, 10 mycelial plugs of these 4 fungi from colony margins were transferred to each of the 6 conical flasks containing 100 mL AEB for shake culture, respectively (25°C, 120 r/min). Azoxystrobin was added to 3 of the 6 conical flasks with the ultimate concentration of 10 g mL-1 36 h later. Two mycelial plugs were taken out from each conical flask after shake culture for 1, 6, 12, 24, 48, and 96 h, one was treated with 150 g mL-1 SHAM and the other one was treated with deionized H2O for 1 h. And then, the ratio of the mycelial oxygen consumption was tested by oxygraph system and the inhibition of respiration on mycelia was calculated.
RESULTS Sensitivity of mycelia and conidia of the 4 fungi to azoxystrobin Test of inhibition on mycelial growth Sensitivity of the 4 fungi to azoxystrobin was determined in vitro. With the concentration of azoxystrobin rising up, the
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inhibition got enhanced and the dose-reaction curve was prominently related. Azoxystrobin had an apparent inhibition on mycelial growth of R. solani and M. grisea with the respective EC50 value 0.008 and 0.013 g mL -1. Inhibitions of azoxystrobin on mycelial growth of C. capsici and B. cinerea were weaker with the respective EC50 values of 10.907 and 70.974 g mL-1. Salicylhydroxamic acid enhanced inhibition of azoxystrobin on mycelial growth. With the application of both azoxystrobin and SHAM, these 4 fungi became more sensitive, especially for C. capsici and R. solani. In the presence of 100 g mL-1 SHAM, the EC50 values of azoxystrobin on R. solani, M. grisea, C. capsici, and B. cinerea were 0.006, 0.007, 0.0268, and 0.329 g mL-1 with the synergistic ratios 1.33, 1.86, 40.40, and 215.73, respectively. Inhibition on conidial germination Azoxystrobin inhibited conidia germination of C. capsici, B. cinerea, and M. grisea with the EC50 values 32.3147, 3.6855, and 0.094 g mL -1. However, the EC 50 values of conidial germination were 0.068, 0.103, and 0.004 g mL-1 in the presence of 100 g mL-1 SHAM with the synergistic ratios 469.24, 35.79, and 23.5, respectively (Table).
Influence of azoxystrobin alone or mixed with SHAM on colony type, conidia type, and pigment biosynthesis Azoxystrobin used alone or mixed with SHAM had different influence on colony type, conidia type, and pigment biosynthesis of the 4 fungi (Fig.1). Once C. capsici was treated with azoxystrobin, the hyphae became sparser and the xanthein was biosynthesized, which could be accelerated by SHAM. Salicylhydroxamic acid alone could not affect pigment biosynthesis. Azoxystrobin inhibited the length and width of the germling tube but did not cause malformation. Azoxystrobin used alone or mixed with SHAM could reduce the airy hyphae of B. cinerea. When B. cinerea was treated with 50 g mL-1 azoxystrobin alone, the airy hyphae were very few. When cooperated with 150 g mL-1 SHAM, only 5 g mL-1 azoxystrobin could inhibit airy hyphae prominently. Azoxystrobin alone could not affect the pigment bio-
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Table EC50 of azoxystrobin alone or with SHAM against mycelial growth and conidial germination of four pathogens Stage
Pathogens
Mycelial growth
Conidial germination
EC50 (g mL-1) - SHAM
Colletotrichum capsici Rhizoctonia solani Magnaporthe grisea Botrytis cinerea Colletotrichum capsici Magnaporthe grisea Botrytis cinerea
EC50 (g mL-1) + SHAM
Factor
0.268 0.006 0.007 0.329 0.068 0.004 0.103
40.40 1.33 1.86 215.73 469.24 23.5 35.79
10.907 0.008 0.013 70.974 31.908 0.094 3.686
(+) and (-) mean present and absent 100 (g mL-1) SHAM, respectively.
A
B CK
0.1 g mL-1
1 g mL-1
CK
0.1 g mL-1 1 g mL-1
10 g mL -1 50 g mL-1 100 g mL-1 10 g mL-1 50 g mL-1 100 g mL-1 CK
0.00625 g mL
-1
C
D 0.00625 g mL-1 0.0125 g mL-1
CK
CK
Azo
SHAM 0.025 g mL-1
0.05 g mL-1
0.1 g mL-1
Fig. 1 Influence of azoxystrobin on the tube characteristics, colony properties, and pigment biosynthesis of the plant pathogens. A, influence of azoxystrobin on germ tub growth of Collethotrichum capsici; B, influence of azoxystrobin used alone (first line) or mixed with SHAM (second line) on the colony properties and pigment biosynthesis of Collethotrichum capsici; C, influence of azoxystrobin and SHAM on the colony properties and pigment biosynthesis of Botrytis cinerea; D, influence of azoxystrobin on the clone properties and pigment biosynthesis of Magnaporthe grisea.
synthesis of B. cinerea but could make the isolate become light brown when cooperated with SHAM. Azoxystrobin used alone or mixed with SHAM could delay the biosynthesis of pigment in M. grisea but hardly affect colony type of R. solani.
Influence on sporulation and sclerotia forming Sporulation of C. capsici was very sensitive to azoxystrobin. The 0.1 g mL-1 azoxystrobin used alone or mixed with 150 g mL-1 SHAM could completely inhibit acervuli from forming. There was no conidiophore production when C. capsici was cultured on plates amended with fungicide even at each concentration after 10 d. Twenty days later, very few (8.7 × 105 conidia
mL -1) conidiophore were produced only in plates amended with 0.1 g mL-1 azoxystrobin alone, which was 99.98% less than the control whose production was 4 × 109 conidia mL-1. In B. cinerea, the production of conidiophore decreased with the concentration of fungicide rising up. The dose-reaction curve was prominently related. When azoxystrobin acted alone, the EC50 value of inhibiting sporulation was 1.1246 g mL-1; while cooperated with SHAM, the EC50 value was 0.0409 g mL-1 at the presence of SHAM. Sclerotia production of R. solani also decreased as the concentration of azoxystrobin rose up. The dosereaction curve was prominently related. The EC50 value of inhibiting sclerotia forming was only 0.0079 g
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Activity of Azoxystrobin and SHAM to Four Phytopathogens
mL-1. When the concentration of azoxystrobin was above 0.5 g mL-1, there was no sclerotia production, either.
Impact of azoxystrobin on mycelial respiration of these 4 fungi Effect of azoxystrobin on mycelial respiration of the 4 fungi of different stages The inhibition effectiveness of azoxystrobin on C. capsici was the best in all of the 4 fungi after 1 h with the inhibition ratio all above 60% in mycelia of different culture time (Fig.2). In contrast, the maximum inhibition was less than 60% for B. cinerea. The inhibition of azoxystrobin on the respiration of mycelia got stronger as the concentration rose up in all 4 fungi. In a word, azoxystrobin had an inhibition on the mycelial respiration of the 4 fungi more or less when they were treated with azoxystrobin for 1 h. Effect of respiration inhibition on mycelia of the 4 fungi by azoxystrobin at different treatment time Azoxystobin alone or mixed with SHAM exhibited different activity against the mycelial respiration of these 4 fungi (Fig.3). The mycelial respira-
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tion of C. capsici and M. grisea were more sensitive to azoxystrobin. As time went on, sensitivity of mycelial respiration of C. capsici and M. grisea to azoxystrobin decreased and was completely lost after 48 and 24 h, respectively. The mycelial respiration of R. solani and B. cinerea were less sensitive to azoxystrobin, and the former showed unchanged sensitivity of mycelial respiration to azoxystrobin, whereas the latter showed similar sensitivity as M. grisea and completely lost the sensitivity of mycelial respiration to azoxystrobin after 12 h. The data on treatment of the inhibitor of alternative oxydase SHAM and its synergistic effects with azoxystrobin indicated that as there were inherent alternative oxidase pathways in R. solani, B. cinerea, and C. capsici, single use of SHAM could inhibit the mycelial respiration of these 3 fungi. But there was no inherent alternative oxidase pathway in M. grisea, single use of SHAM showed no inhibitory activity against mycelial respiration of this fungus. However, alternative oxidase pathway appeared after 12 h treatment with azoxystrobin and strengthened as the time went on, indicating that alternative oxidase pathway could be induced by azoxystrobin in M. grisea.
Fig. 2 Inhibition of oxygen consumption in 4 fungal mycelia by azoxystrobin. A, inhibition of oxygen consumption in Colletotrichun capsici in different aged mycelia by azoxystrobin at different concentrations; B, inhibition of oxygen consumption in Rhizoctonia solani in different aged mycelia by azoxystrobin at different concentrations; C, inhibition of oxygen consumption in Magnaporthe grisea in different aged mycelia by azoxystrobin at different concentrations; D, inhibition of oxygen consumption in Botrytis cinerea in different aged mycelia by azoxystrobin at different concentrations.
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Fig. 3 Inhibition of oxygen consumption in 4 fungal mycelia treated with azoxystrobin or azoxstrobin plus SHAM at different time. A, inhibition of oxygen consumption in Colletotrichun capsici; B, inhibition of oxygen consumption in Rhizoctonia solani; C, inhibition of oxygen consumption in Magnaporthe grisea; D, inhibition of oxygen consumption in Botrytis cinerea.
DISCUSSION Since Zeneca Company (UK) developed the first commercial fungicide of strobilurins in 1996, the researches in this class of fungicides were extensive. It was reported that the spores of C. capsici were more sensitive to azoxystrobin than that in mycelia; and moreover, SHAM exhibited synergistic effects on inhibiting conidial germination but did not exhibit synergistic effects in inhibiting mycelial growth (Li et al. 2005). This study also showed that the spores of C. capsici were more sensitive to azoxystrobin than that in mycelia; however, SHAM exhibited synergistic effects on inhibiting both conidial germination and mycelial growth. Sierotaki et al. (2000) also found that trifloxystrobin mixed with SHAM had a better inhibition on the mycelial growth of Mycosphaerella fijiensis than trifloxystrobin alone. Karadimosa et al. (2005) also found that Cercospora beticola were more sensitive to trifloxystrobin than its mycelia. Mizutani et al. (1995, 1996) found that when M. grisea was treated with metominostrobin, the content of active oxygen species increased and the activity of alternative oxidase got stronger, and SHAM enhanced the effectiveness of azoxystrobin by preventing the alternative oxidase pathway from induced. Previous studies showed that on the plates of me-
dium for M. grisea to produce spores amended with 0.01 g mL-1 azoxystrobin, the spore production was 1.57 × 106 spores per plate, decreasing by 43.7% when compared with the nonamended control and the pigment biosynthesis was inhibited (Zhang et al. 2005). In this study, although spore germination of M. grisea was strongly inhibited by azoxystrobin (similar to the previous results), the pigment biosynthesis of M. grisea was only delayed by azoxystrobin. Besides inhibiting the mycelial growth and conidial germination, azoxystrobin with/without SHAM could also affect the conidia and sclerotia production of these pathogens. So it could play an important role during the whole life cycles of many pathogens and could resist pathogens from infecting and expanding if the hosts had already been infected. It could also reduce the offspring and the reinfection source so as to control the diseases effectively. Therefore, when azoxystrobin was protectively used in the field, plant antioxidants present in the host quenched the active oxygen species from reaching the level sufficient for inducing alternative oxidase pathway so as to control the plant diseases effectively. And it was much better to add SHAM according to the field conditions during the in vitro test. Also, it was suggested to protectively use azoxystrobin so as to control the plant diseases effectively based on our results. Moreover, Avila-Adame and Köller (2003)
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Activity of Azoxystrobin and SHAM to Four Phytopathogens
found that protective use of azoxystrobin could not only better control the plant disease (than curative use of azoxystrobin) but also prevent the alternative oxidase pathway during conidia germination so as to delay the development of fungicide resistance. Oxygen consumption test of mycelia in this essay showed that azoxystrobin inhibited mycelial respiration of these 4 pathogens to different extent in the early stages and the inhibition on mycelia of C. capsici was the strongest. And the inhibition effectiveness became better with the concentration of azoxystrobin rising up. However, as time went on, the respiration of the mycelia treated with azoxystrobin got stronger and SHAM could not reduce the oxygen consumption. The authors speculated that the strengthened expression of cyt bc1 complex genes might make the fungi insensitive to the fungicide. As reported by Bourges et al. (2005), the QoI inhibitor myxothiazol could change the expression of many genes, such as cytoplast catalase gene, the transformation factor Yap1, and so on. Whether this was caused by the strengthened expression of cyt bc1 complex genes should be further explored. And moreover, recently, Chen et al. (2009) found that the enhanced cyt b gene expression might play a role in the development of resistance to azoxystrobin in C. capsici. Results above also indicated that mycelia respiration had indirect relationship with mycelia growth. Mizutani et al. (1995) also reported that asexual growth hardly depended on respiration. Thus, the authors also speculated that azoxystrobin might affect other metabolisms except energy system, which is also needed to be further studied.
CONCLUSION This study presented the effect of single use of azoxystrobin or with SHAM on the sensitivity, biological properties, sporulation, or sclerotia forming, and mycelial respiration of the 4 fungi. Some conclusions could be drawn as belows: (1) Azoxystrobin could inhibit the mycelial growth of all of the 4 fungi, and the conidial germination of three of the 4 fungi. And SHAM exhibited synergistic effects with azoxystrobin. (2) Azoxystrobin affected the colony type and conidia production of C. capsici and B. cinerea, sclerotia pro-
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duction of R. solani, prevented the pigment biosynthesis of C. capsici, and delayed pigment biosynthesis of M. grisea. (3) Single use of azoxystrobin could inhibit the mycelial respiration of all the 4 fungi. The inhibition increased as the concentration increased. And as the time went on, the inhibition of azoxystrobin decreased and disappeared in these fungi except R. solani. (4) SHAM showed little synergistic effects against mycelial respiration of these fungi. The expression of alternative oxidase pathway was induced by 12 h treatment of azoxystrobin in M. grisea; however, alternative oxidase pathway was constitutively expressed in other 3 fungi.
Acknowledgements This study was sponsored by the National 973 Program of China (2009CB118906, 2006CB101907), the National 863 Program of China (2006AA10A211, 2008AA10Z414), and the National Natural Science Foundation of China (30671048, 30671384).
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