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Effects of some environmental conditions on the effectiveness of Drechslera avenacea (Curtis ex Cooke) Shoem.: a potential bioherbicidal organism for Avena fatua L.
Biological Control 24 (2002) 103–109 www.academicpress.com
Effects of some environmental conditions on the effectiveness of Drechslera avenacea (Curtis ex Cooke) Shoem.: a potential bioherbicidal organism for Avena fatua L. Shane D. Hetherington,* Heather E. Smith, Melanie G. Scanes, and Bruce A. Auld NSW Agriculture, Orange Agricultural Institute, Forest Road, Orange, NSW, Australia Received 10 October 2000; accepted 31 December 2000
Abstract Drechslera avenacea is a potential bioherbicide for Avena fatua, wild oat, control in dryland wheat crops in southern Australia. Maximum disease severity (DS) (1.1 lesions per mm2 of leaf tissue) was recorded following application of 1 105 spores per ml and exposure of weeds to a 12- to 16-h dew period at 20–25 °C. Weed seedlings between 3 and 5 weeks of age were the most susceptible to the disease. Occurrence of a low humidity ð< 50% R.H.) for up to 8-h before the initiation of a dew period following inoculation did not reduce subsequent DS. Repeated 8-h (suboptimal) dew periods increased DS when compared with a single 8-h dew period (0.26 lesions per mm2 compared to 0.39 lesions per mm2 , respectively). This was not the case for repeated 6-h dew periods. Following inoculation, temperature had a direct effect on disease development rate. Plants recovered following inoculation and inoculated and uninoculated plants were not significantly different in terms of necrotic leaves 28 days after inoculation. Dew period limitations and the ability of D. avenacea to indirectly create a competitive advantage for wheat seedlings over wild oat are discussed in relation to the feasibility of this bioherbicide agent. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Drechslera avenacea; Avena fatua; Bioherbicide; Etiology; Dew period; Plant age
1. Introduction Despite multi-faceted control strategies, Avena fatua L. (wild oat) remains one of the world’s worst weeds (Holm et al., 1977). Herbicides are widely used against this weed and provide selective control. However, herbicide-resistant populations have been recorded in Europe, North and South Americas, and Australia. In Australia there is evidence to suggest that populations of herbicide-resistant wild oat are increasing in size (Pratley, 1996). Alternative methods are being investigated as a means of reducing evolutionary selection pressure toward resistance (Thill et al., 1994). Because the weed is closely related to cultivated oat (Avena sativa L.) classical biological control involving the introduction of exotic organisms is considered *
Fax: +61-2-63913899. E-mail address: [email protected] (S.D. Hetherington).
unfeasible. The inundative or bioherbicidal approach involving a targeted release of indigenous pathogens is more appropriate. Several projects have targeted A. fatua. Wilson (1987) concluded that the fungal pathogen Drechslera avenae (Eidam) Scharif. (a synonym of D. avenacea; (Sivanesan, 1987)) was unsuitable for use as a mycoherbicide against wild oat because of a combination of low spore production, low virulence, infection of some crop plants, and a requirement for temperatures and moisture not routinely encountered in the field. Similar studies are being carried out by Zonjian and Yanghan (1996) in China examining the pathogen Drechslera avenaceae (sic) (Curtis and Cooke) Shoem. Surveys done in New South Wales, Victoria, and Western Australia representing wheat-growing and coastal regions where wild oat is present yielded a highly virulent and host-selective isolate of D. avenacea (Hetherington et al., 1998). This fungus causes a foliar disease which, when severe, produces red-black lesions
1049-9644/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 9 - 9 6 4 4 ( 0 2 ) 0 0 0 2 0 - 8
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that expand and coalesce resulting in broad foliar necrosis. Isolates of D. avenacea were subsequently collected from diverse geographical locations representing the environmental limits of this fungus. Some 400 isolates of the species were grown as monoconidial cultures and screened in preliminary trials. The use of this organism as the basis of a mycoherbicide requires that its disease etiology be well understood to help overcome some of the perceived shortcomings of this pathogen encountered by earlier workers. The objective of this study was to examine the temperature and dew requirements of the fungus to cause disease, and the response of the host to infection.
2. Materials and methods 2.1. Seed source and plant growth Seed of naturalized A. fatua was collected from the field in central western New South Wales. Plants were grown in a steam-pasteurized sand-peat (3:1) mix in 10-cm-diam pots. 2.2. Fungal inoculum D. avenacea was isolated from diseased plants growing near Mathoura, NSW, Australia. An isolate (IMI375958) was chosen as preliminary testing indicated that it had a shorter dew-period requirement than other isolates (a desirable trait for a mycoherbicidal fungus). The isolate was single-spored, cultured, lyophilized, and stored at room temperature until required. When required, the fungus was rehydrated and grown on green bean agar (GBA; Wilson, 1987) for 7 days. Plates were then flooded with 10 ml of sterile distilled water and the surface of fungal colonies scraped with the edge of a flamed microscope slide. The resultant fungal suspension was sequentially poured onto the surface of a number of GBA agar plates and the colonies allowed to grow for 10 days. These colonies were then flooded with 5–10 ml of sterile distilled water and the resulting suspension filtered through two layers of sterile cheesecloth to exclude mycelium. The conidial concentration was measured using a haemocytometer and standardized to the required concentration by the addition of sterile distilled water. All conidial suspensions were amended with 100 ll of Tween 20 (polyoxyethylenesorbitan monolaurate; Sigma Aldrich, Australia) per 100 ml of inoculum. Plants were inoculated using a small, pressurized spray gun at 200 kpa (Puma, model no. AS1010, Taiwan). For this and all other experiments, a petri dish containing water agar was sprayed with the inoculum, and spore viability assessed 24 h after inoculation by counting germinated and ungerminated conidia.
2.3. Plant age and susceptibility Twenty pots (10-cm diam; 9.5-cm height) of A. fatua (2 seedlings per pot ¼ 1 replicate) were planted at weekly intervals for 6 weeks. The youngest seedlings were allowed to grow to first true leaf stage (Zadoks’ 11; Zadoks et al., 1974) before 10 plants of each age were inoculated with a conidial suspension at a concentration of 1 105 conidia per ml. The remaining plants were sprayed with water and Tween 20. All plants were then placed in a dew chamber at 20 °C for 16-h before being transferred to a controlled environment (CE) cabinet kept at 20 °C with a 12-h light/dark photocycle for 10 days. At this time, the above-ground portion of plants was removed and placed in an oven (80 °C) overnight and their dry weights measured. An index of disease severity was generated by subtracting the dry weights of inoculated plants from uninoculated controls for each treatment. Because of large weight differences between seedlings of different ages the disease severity index was transformed ðlog þ0:1Þ to allow inter-treatment comparison. 2.4. Dew-period temperature Sixty-five pots of A. fatua seedlings (2 per pot ¼ 1 replicate) were grown to 3-leaf stage (Zadoks’ 13) and 60 of these were inoculated with a conidial suspension of D. avenacea at a concentration of 5 104 conidia per ml to the point of run-off. Five pots were sprayed with water and Tween 20 only. Fifty of the inoculated pots were placed in a dew chamber (Percival, Boone, Iowa) at 10 °C under darkness. The remaining 15 pots were placed in a dark CE cabinet at 20 °C. Ten plants were transferred from the dew chamber to the CE cabinet 4, 8, 12, 16, and 24 h after inoculation. Following the final transfer, lights were turned on inside the CE cabinet and set for a 12-h light/dark photocycle. When lesions became visible (approx. 2 days after inoculation), the number of lesions per mm2 of leaf surface was assessed; 4 days after inoculation, the number of necrotic and healthy leaves was counted. The above-mentioned experimental procedure was repeated with the dewchamber temperatures maintained at 12, 15, 20, and 25 °C. 2.5. Delayed dew periods Sixty pots of A. fatua seedlings (2 per pot ¼ 1 replicate) were grown to 3-leaf stage (Zadoks’ 13) and inoculated with a D. avenacea conidial suspension at a concentration of 5 104 conidia per ml. Seedlings were then subjected to the following dew-period treatments, all in the dark: 16-h dew period, 12 h in CE cabinet; 1 h in CE cabinet, 16-h dew period, 11 h in CE cabinet; 2 h in CE cabinet, 16-h dew period, 10 h in CE cabinet;
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4 h in CE cabinet, 16-h dew period, 8 h in CE cabinet; 6 h in CE cabinet, 16-h dew period, 6 h in CE cabinet; and 8 h in CE cabinet, 16-h dew period, 4 h in CE cabinet. Following the final transfer, lights were turned on inside the CE cabinet and set for a 12-h light/dark photocycle. When lesions were visible to the unaided eye (approx. 2 days after inoculation), the number of lesions per mm2 of leaf surface was assessed using a stereo dissecting microscope. Four days after inoculation, the number of necrotic and healthy leaves was counted. Treatment means were compared using the Tukey’s x procedure. 2.6. Repeated suboptimal dew periods Forty pots of A. fatua seedlings (2 per pot ¼ 1 replicate) were grown to 3-leaf stage (Zadoks’ 13) and inoculated with a D. avenacea conidial suspension at a concentration of 5 104 conidia per ml. Seedlings were then subjected to the following dew-period treatments, all in the dark: 6-h dew period, 22-h CE cabinet; 8-h dew period, 20-h CE cabinet; 6-h dew period, 12-h CE cabinet, 6-h dew period, 4-h CE cabinet; and 8-h dew period, 12-h CE cabinet, 8-h dew period. Humidity within the CE cabinet was maintained below 50% R.H. and the temperature was 25 °C. Following the final transfer, lights were turned on inside the CE cabinet set for a 12-h light/dark photocycle. When lesions were visible to the unaided eye (approx. 2 days after inoculation), the number of lesions per mm2 of leaf surface was assessed using a stereo dissecting microscope. Four days after inoculation, the number of necrotic and healthy leaves was counted.
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dew chamber at 20 °C under darkness for a period of 16 h and then placed in a CE cabinet at 25 °C. For five pots from each spore concentration, the number of lesions per mm2 of leaf area was assessed 4 days after inoculation. The remaining seedlings were allowed to grow for a further 6 days (a total of 10 days after inoculation) before the proportion of dead leaves was assessed and dry weights recorded. 2.9. Vegetative growth recovery Sixty pots of A. fatua seedlings (2 per pot ¼ 1 replicate) were grown to 3-leaf stage (Zadoks’ 13) and inoculated with a D. avenacea conidial suspension at a concentration of 5 104 conidia per ml. Ten of these plants were placed in a dark CE cabinet at 20 °C. The remaining plants were placed in a dew chamber at 25 °C with 10 plants being transferred to the CE cabinet 4, 8, 12, 16, and 24 h after inoculation. Following the final transfer, the lights in the CE cabinet were turned on and the plants subjected to a 12-h dark/light photocycle. After 4 days, the number of necrotic and healthy leaves on each plant was assessed. Leaves were classified as dead when greater than 80% of their tissue was necrotic. This assessment was carried out at weekly intervals for the following 3 weeks. 2.10. Statistical analysis All analyses, except where noted, were undertaken using the Genstat statistical analysis system (IACRRothamsted, Harpenden, Herts, England).
3. Results 2.7. Post-inoculation temperature 3.1. Plant age and susceptibility Two A. fatua seedlings were grown in 50, 10-cm-diam pots. When seedlings had matured to the 3-leaf stage (Zadoks’ 13) they were inoculated with a spore suspension at a concentration of 5 104 conidia per ml and placed in a dew chamber for 16-h at 15 °C under darkness. Ten seedlings were then placed in CE cabinets at 10, 15, 20, 25, and 30 °C. When lesions appeared, the number of lesions per mm2 was assessed for each treatment. The percentage of leaves with > 80% necrotic tissue was assessed 10 days after inoculation. 2.8. Inoculum concentration Sixty pots of A. fatua seedlings (2 per pot ¼ 1 replicate) were grown to the 3-leaf stage (Zadoks’ 13). Ten plants were inoculated with a spore suspension of 1 104 , 5 104 , 1 105 ; 2 105 , and 3 105 spores per ml of D. avenacea. Ten pots of seedlings were sprayed with water and Tween 20 only. All plants were placed in a
To compensate for the lower dry weight of young plants compared to older plants, data were log-transformed before analysis. Young seedlings (1- and 2-week postemergence) were less susceptible to D. avenacea than older seedlings. Disease severity was high when plants were from 3 to 6 weeks of age and was maximal when plants were inoculated approximately 4ð1=2Þ weeks after emergence (Fig. 1). Plants were highly susceptible when inoculated at 4 and 5 weeks postemergence. Over 50% of the total tissue was necrotic in plants inoculated at 3-, 4-, and 5-week postemergence. 3.2. Dew-period temperature It was not possible to examine dew period responses at temperatures greater than 30 °C as seedlings began to wilt and necrose independent of their inoculation state. A regression equation describing the interaction be-
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3.3. Delayed dew periods A delay of up to 8 h following inoculation did not reduce disease severity (P < 0:005; data not shown). 3.4. Repeated suboptimal dew periods
Fig. 1. Susceptibility of A. fatua seedlings of various ages to D. avenacea. Dry weights of seedlings inoculated with a conidial suspension of D. avenacea ð1 105 conidia=mlÞ and uninoculated controls were recorded 10 days after inoculation. Relative disease severity is the natural log þ0:1 of differences between the mean dry weights ðn ¼ 5Þ of uninoculated control plants and inoculated plants of varying ages. Data points ð Þ are the means of treatments. The regression y ¼ 0:1278x2 þ 1:183 3:341 (—) accounted for 92.3% of treatment variance.
tween dew period, temperature, and disease severity was generated using SigmaStat (Jandel, CA, USA). Dew period, the temperature during the dew period, and the interaction of these two variables had significant effects ðP < 0:05Þ on disease severity. The optimum temperature range for maximum disease severity was 20–25 °C, which was more pronounced as dew period increased (Fig. 2).
Fig. 2. The effect of post-inoculation dew period and temperature upon disease severity of A. fatua seedlings by D. avenacea. The regression z ¼ 1:2e ½ððx 22:4Þ=7:2Þ2 þ ððy 20:2Þ=7:9Þ2 was fitted to the data (r2 ¼ 0.88).
Due to its skewed nature, the data set was logtransformed. An unpaired t test was used to compare 6-h dew period with 6-h repeated dew-period treatments and 8-h dew period with 8-h repeated dew-period treatments. Following a 6-h dew period, an average of 0.29 lesions per mm2 of leaf tissue was recorded. Subjecting plants to a second 6-h dew period following a 12-h low humidity period (average 0.26 lesions per mm2 ) did not increase this disease severity. Following a dew period of 8-h, 0.26 lesions per mm2 formed. A second 8-h dew period following a 12-h delay resulted in an increase in disease severity (0.39 lesions per mm2 ; P < 0:05). 3.5. Post-inoculation temperature Lesions on plants incubated at higher temperatures following inoculation expanded more rapidly and coalesced resulting in large necrotic areas and finally entirely necrosed leaves. There was a linear relationship between post-inoculation temperature and disease development (Fig. 3). 3.6. Inoculum concentration Data were not distributed normally within treatments and therefore a log-transformation was undertaken before analysis. An exponential model was fitted to the
Fig. 3. The effect of temperature on disease severity following infection of A. fatua seedlings by D. avenacea. Data points ð Þ represent treatment means. The regression y ¼ 2:5 23 (—) accounted for 70.5% of treatment variance.
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4. Discussion
Fig. 4. The effect of D. avenacea inoculum concentration (conidia/ml) on the severity of disease on A. fatua seedlings. Data points ð Þ represent treatment means. The regression y ¼ 0:3 0:3ð0:9999813x Þ accounted for 83.7% of treatment variance.
data and accounted for 80% of the variance (Fig. 4). Disease severity was affected by the inoculum concentration applied to the seedlings (P < 0:05). Disease levels rose sharply as inoculum concentration was increased to 1 105 conidia per ml. Increased concentration beyond this point did not result in large increases in disease severity. 3.7. Vegetative growth recovery At 7, 14, and 21 days after inoculation, all treatments except the 4-h dew-period treatment resulted in greater necrosis than uninoculated controls (Fig. 5). Twentyeight days after inoculation there was no difference ðP < 0:05Þ in the percentage of dead leaves between treatments.
Fig. 5. Recovery in terms of vegetative growth of A. fatua following inoculation of seedlings with a conidial suspension of D. avenacea at a concentration of 1 105 conidia/ml.
An appreciation of the biology of a weed, its potential biological control agent, and their interaction within their environment is an essential prerequisite to the initiation of a biological weed control program (Wapshere, 1975). Where the biological control program intends the use of a mycoherbicide, a number of these biological parameters have often constituted constraints to the success of previous systems (Auld and Morin, 1995). Central to these parameters are the innate virulence of the pathogen and its ability to initiate disease under limiting moisture conditions. In Australia, wheat, the primary market for any chemical or biological herbicide intended for wild oat, is grown under dryland conditions and is dependent on natural rainfall. Rainfall is highest in winter in the wheat-growing area although somewhat erratic in occurrence. Wild oat control is required early in the cropping cycle, generally before the crop reaches tillering (Mullen et al., 2000). This is also the only time at which D. avenacea produces a significant impact on wild oat growth. Our data suggest that a window of opportunity exists for the optimum use of D. avenacea to control wild oat when seedlings are between 3 and 5 weeks postemergence (Fig. 1). No reliable data exist on dew-period lengths in the Australian wheat belt but they are thought to be typically under 12 h. Planting occurs in late autumn or early winter and therefore D. avenacea must be applied during early to mid-winter. Average minimum temperatures in southern NSW for this period are 2–8 °C (MetAccess (CSIRO, 1999) data for Wyalong [33°560 S; 147°160 E] 1989–1999 May, June, July). Because of low temperatures at the time of application of D. avenacea, it is important that the interaction between dew period dependence and temperature be studied. We found that the optimum temperature for maximum disease development was 20–25 °C. At 10 °C only minimal disease was recorded for dew periods less than 12 h (Fig. 2). In the absence of suitable periods of overnight dew, bioherbicide application strategies might also take advantage of free water on the leaf surface provided by rain. This water would be less predictable and the agent may need to withstand delays before the initiation of dew or repeated suboptimal moisture periods. D. avenacea remains viable and capable of causing severe disease on the leaf surface for up to 8 h following application. The severity of disease recorded following an 8-h dew period was increased by a second 8-h dew period 12 h after the first. However two 6-h dew periods did not improve the pathogens efficacy. Hence, D. avenacea is not totally reliant upon overnight dew to initiate disease but would also respond to rainfall or mist occurring at appropriate times. These events may occur during the day when temperature is more likely to
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favor infection. In practice, efficacy would depend on the occurrence of appropriate environmental conditions and would require immediate response from wheat farmers. The post-inoculation temperature also has an effect on disease severity. We assessed leaf necrosis as an index to disease severity in this case as it gives an indication of the rate of disease spread rather than the infection rate per se. It was noted that lesions spread and coalesced resulting in wholly necrotic leaves more rapidly at higher temperatures. This resulted in a linear relationship between post-infection temperature and disease severity (Fig. 3). Again this would result in the efficacy of a product based on this organism being subject to environmental fluctuation. In a similar study, following the infection of malesterile hybrids of maize in the United States of America, Bipolaris maydis (Nisikado and Miyake) Shoem., a pathogen closely related to D. avenacea was found to be ‘‘not an effective cool weather parasite’’ (Nelson and Tung, 1972). Disease development following infection by B. maydis was favored by dew periods of 12 h or more, dew-period temperatures of 24 °C or more, and high post-dew-period temperatures. The authors suggested that disease epidemics in cooler maize-producing regions in the USA failed to become established because of the temperature dependence of the pathogen. Similarly, because of its dependence on high temperatures for infection, D. avenacea may be more useful as a bioherbicide in warmer, northern wheat-growing regions in Australia. Given an ideal environment, the severity of disease approaches a maximum following the application of inoculum at a concentration of 1 105 conidia per ml (Fig. 4). At this and higher concentrations, total necrosis of all inoculated leaves often occurs. However, regrowth from enclosed meristems occurs quickly. Four weeks after inoculation, recovery is complete and there is no difference between inoculated and uninoculated control plants (Fig. 5). Were this to occur in the field, it may provide a competitive advantage for wheat seedlings over wild oat during a critical phase in the crop cycle, similar to that reported for competitive cultivars (Lemerle and Cousens, 1992; Seavers and Wright, 1999). Cultivars with higher growth rates in leaf and tiller production have an advantage over other grass species. Modeling has indicated that employment of competitive cultivars may lead to a reduction in the rate of build up of herbicide resistance by up to 50% (Gill, 1998) and therefore similar outcomes may result from the development and deployment of D. avenacea as a bioherbicide to control wild oat. Strategic application of D. avenacea as a biological control agent and the consequent weakening of A. fatua may have a place in integrated weed control, particularly given the spread of herbicide resistance. Two major
problems exist. First the dew period requirement for this pathogen is unrealistic at low temperature and tactical application to take advantage of unpredictable rainfall is not a practical option. Second, D. avenacea does not produce spores in submerged liquid culture as generally preferred for large-scale commercial production of bioherbicides (Auld and Morin, 1995). Generic formulation research underway in our laboratory may provide a solution to the first of these problems. The use of solid-state fermentation systems for the production of biological control agents is also an area of active research (Ooijkaas et al., 2000). Bioherbicides continue to suffer through direct comparison of their efficacy to chemical herbicides (Auld and Morin, 1995). An herbicide based on D. avenacea will not provide the efficacy in terms of mortality that a chemical herbicide may. However, it has the potential through infection and growth retardation of weed seedlings to provide a competitive advantage to crop seedlings. Additionally, it is a fairly specific pathogen (Hetherington and Auld, 2001), and pending technical solutions to water requirement and production constraints, remains a candidate as a biological control agent of wild oat.
Acknowledgment The authors wish to acknowledge the financial support of the Australian Centre for International Agricultural Research for providing funding to conduct the research.
References Auld, B.A., Morin, L., 1995. Constraints in the development of bioherbicides. Weed Technol. 9, 638–652. CSIRO., 1999. Metaccess. Analysing Weather Record, Version 1.04. Horizon Technology Pty. Ltd., Roseville, NSW, Australia. Gill, G., 1998. Reducing herbicide use: the role of competitive cultivars. Final Report, Grains Research and Development Corporation, Australia. Hetherington, S.D., Auld, B.A., 2001. Host range of Drechslera avenacea, a fungus with potential for use as a biological control agent of Avena fatua. Aust. Plant Pathol. 30, 205–210. Hetherington, S.D., Auld, B.A., Smith, H.E., 1998. A possible bioherbicide for Avena fatua L. (wild oats): isolate collection and host range testing. Proc. Linnaean Soc. NSW 119, 219–225. Holm, L.G., Plucknett, D.L., Pancho, J.V., Herberger, J.P., 1977. The World’s Worst Weeds: Distribution and Biology. University Press of Hawaii, Honolulu, HI. Lemerle, D., Cousens, R.D., 1992. Suppression of weeds by competitive wheat cultivars and interaction with herbicide. In: Combellack, J.H. (Ed.), Proceedings of the 1st International Weed Control Congress. Weed Science Society, Melbourne, Australia, pp. 282– 284. Mullen, C.L., Dellow, J.J., Tonkin, C.J., 2000. Using the growth stages of cereal crops to time herbicide application. In: Anon (Ed.), Weed
S.D. Hetherington et al. / Biological Control 24 (2002) 103–109 Control In Winter Crops, NSW Agriculture, Sidney, Australia, p. 7. Nelson, R.R., Tung, G., 1972. Effect of dew temperature, dew period, and post dew temperature on infection of a male-sterile corn hybrid by race T of Helminthosporium maydis. Plant Dis. Rep. 56, 767–769. Ooijkaas, L.P., Weber, F.J., Buitelaar, R.M., Tramper, J., Rinzema, A., 2000. Defined media and inert supports: their potential as solidstate fermentation production systems. Trends Biotechnol. 18, 356– 360. Pratley, J.E., 1996. Herbicide Resistance in Southern New South Wales. Final Report, Grains Research and Development Corporation, Kingston, ACT, Australia. Seavers, G.P., Wright, K.J., 1999. Crop canopy development and structure influence weed suppression. Weed Res. 39, 319–328. Sivanesan, A., 1987. Graminicolous Species of Bipolaris, Curvularia, Drechslera, Exserohilum, and their Teleomorphs. C.A.B. International, Wallingford, Oxon, UK.
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Thill, D.C., O’Donovan, J.T., Mallory-Smith, C.A., 1994. Integrated weed management strategies for delaying herbicide resistance in wild oats. Phytoprotection 75, 61–70. Wapshere, A.J., 1975. A protocol for biological control of weeds. PANS 21, 295–303. Wilson, S., 1987. Scope for biological control of Avena fatua L. with Drechslera avenae (Eidam) Sharif. Ph.D. Thesis, Department of Plant Sciences, Oxford University, UK. Zadoks, J.C., Chang, T.T., Conzak, C.F., 1974. A decimal code for the growth stages of cereals. Weed Res. 14, 415–421. Zonjian, Z., Yanghan, L., 1996. Discovery, isolation and pathogenicity study of a wild oat (Avena fatua) biological control fungus. In: Anon. (Ed.), Proceedings of the 3rd International Bioherbicide Workshop—Programme and Abstracts. International Bioherbicide Group, Stellenbosch, South Africa, p. 30.
Effects of some environmental conditions on the effectiveness of Drechslera avenacea (Curtis ex Cooke) Shoem.: a potential bioherbicidal organism for Avena fatua L. Shane D. Hetherington,* Heather E. Smith, Melanie G. Scanes, and Bruce A. Auld NSW Agriculture, Orange Agricultural Institute, Forest Road, Orange, NSW, Australia Received 10 October 2000; accepted 31 December 2000
Abstract Drechslera avenacea is a potential bioherbicide for Avena fatua, wild oat, control in dryland wheat crops in southern Australia. Maximum disease severity (DS) (1.1 lesions per mm2 of leaf tissue) was recorded following application of 1 105 spores per ml and exposure of weeds to a 12- to 16-h dew period at 20–25 °C. Weed seedlings between 3 and 5 weeks of age were the most susceptible to the disease. Occurrence of a low humidity ð< 50% R.H.) for up to 8-h before the initiation of a dew period following inoculation did not reduce subsequent DS. Repeated 8-h (suboptimal) dew periods increased DS when compared with a single 8-h dew period (0.26 lesions per mm2 compared to 0.39 lesions per mm2 , respectively). This was not the case for repeated 6-h dew periods. Following inoculation, temperature had a direct effect on disease development rate. Plants recovered following inoculation and inoculated and uninoculated plants were not significantly different in terms of necrotic leaves 28 days after inoculation. Dew period limitations and the ability of D. avenacea to indirectly create a competitive advantage for wheat seedlings over wild oat are discussed in relation to the feasibility of this bioherbicide agent. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Drechslera avenacea; Avena fatua; Bioherbicide; Etiology; Dew period; Plant age
1. Introduction Despite multi-faceted control strategies, Avena fatua L. (wild oat) remains one of the world’s worst weeds (Holm et al., 1977). Herbicides are widely used against this weed and provide selective control. However, herbicide-resistant populations have been recorded in Europe, North and South Americas, and Australia. In Australia there is evidence to suggest that populations of herbicide-resistant wild oat are increasing in size (Pratley, 1996). Alternative methods are being investigated as a means of reducing evolutionary selection pressure toward resistance (Thill et al., 1994). Because the weed is closely related to cultivated oat (Avena sativa L.) classical biological control involving the introduction of exotic organisms is considered *
Fax: +61-2-63913899. E-mail address: [email protected] (S.D. Hetherington).
unfeasible. The inundative or bioherbicidal approach involving a targeted release of indigenous pathogens is more appropriate. Several projects have targeted A. fatua. Wilson (1987) concluded that the fungal pathogen Drechslera avenae (Eidam) Scharif. (a synonym of D. avenacea; (Sivanesan, 1987)) was unsuitable for use as a mycoherbicide against wild oat because of a combination of low spore production, low virulence, infection of some crop plants, and a requirement for temperatures and moisture not routinely encountered in the field. Similar studies are being carried out by Zonjian and Yanghan (1996) in China examining the pathogen Drechslera avenaceae (sic) (Curtis and Cooke) Shoem. Surveys done in New South Wales, Victoria, and Western Australia representing wheat-growing and coastal regions where wild oat is present yielded a highly virulent and host-selective isolate of D. avenacea (Hetherington et al., 1998). This fungus causes a foliar disease which, when severe, produces red-black lesions
1049-9644/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 9 - 9 6 4 4 ( 0 2 ) 0 0 0 2 0 - 8
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that expand and coalesce resulting in broad foliar necrosis. Isolates of D. avenacea were subsequently collected from diverse geographical locations representing the environmental limits of this fungus. Some 400 isolates of the species were grown as monoconidial cultures and screened in preliminary trials. The use of this organism as the basis of a mycoherbicide requires that its disease etiology be well understood to help overcome some of the perceived shortcomings of this pathogen encountered by earlier workers. The objective of this study was to examine the temperature and dew requirements of the fungus to cause disease, and the response of the host to infection.
2. Materials and methods 2.1. Seed source and plant growth Seed of naturalized A. fatua was collected from the field in central western New South Wales. Plants were grown in a steam-pasteurized sand-peat (3:1) mix in 10-cm-diam pots. 2.2. Fungal inoculum D. avenacea was isolated from diseased plants growing near Mathoura, NSW, Australia. An isolate (IMI375958) was chosen as preliminary testing indicated that it had a shorter dew-period requirement than other isolates (a desirable trait for a mycoherbicidal fungus). The isolate was single-spored, cultured, lyophilized, and stored at room temperature until required. When required, the fungus was rehydrated and grown on green bean agar (GBA; Wilson, 1987) for 7 days. Plates were then flooded with 10 ml of sterile distilled water and the surface of fungal colonies scraped with the edge of a flamed microscope slide. The resultant fungal suspension was sequentially poured onto the surface of a number of GBA agar plates and the colonies allowed to grow for 10 days. These colonies were then flooded with 5–10 ml of sterile distilled water and the resulting suspension filtered through two layers of sterile cheesecloth to exclude mycelium. The conidial concentration was measured using a haemocytometer and standardized to the required concentration by the addition of sterile distilled water. All conidial suspensions were amended with 100 ll of Tween 20 (polyoxyethylenesorbitan monolaurate; Sigma Aldrich, Australia) per 100 ml of inoculum. Plants were inoculated using a small, pressurized spray gun at 200 kpa (Puma, model no. AS1010, Taiwan). For this and all other experiments, a petri dish containing water agar was sprayed with the inoculum, and spore viability assessed 24 h after inoculation by counting germinated and ungerminated conidia.
2.3. Plant age and susceptibility Twenty pots (10-cm diam; 9.5-cm height) of A. fatua (2 seedlings per pot ¼ 1 replicate) were planted at weekly intervals for 6 weeks. The youngest seedlings were allowed to grow to first true leaf stage (Zadoks’ 11; Zadoks et al., 1974) before 10 plants of each age were inoculated with a conidial suspension at a concentration of 1 105 conidia per ml. The remaining plants were sprayed with water and Tween 20. All plants were then placed in a dew chamber at 20 °C for 16-h before being transferred to a controlled environment (CE) cabinet kept at 20 °C with a 12-h light/dark photocycle for 10 days. At this time, the above-ground portion of plants was removed and placed in an oven (80 °C) overnight and their dry weights measured. An index of disease severity was generated by subtracting the dry weights of inoculated plants from uninoculated controls for each treatment. Because of large weight differences between seedlings of different ages the disease severity index was transformed ðlog þ0:1Þ to allow inter-treatment comparison. 2.4. Dew-period temperature Sixty-five pots of A. fatua seedlings (2 per pot ¼ 1 replicate) were grown to 3-leaf stage (Zadoks’ 13) and 60 of these were inoculated with a conidial suspension of D. avenacea at a concentration of 5 104 conidia per ml to the point of run-off. Five pots were sprayed with water and Tween 20 only. Fifty of the inoculated pots were placed in a dew chamber (Percival, Boone, Iowa) at 10 °C under darkness. The remaining 15 pots were placed in a dark CE cabinet at 20 °C. Ten plants were transferred from the dew chamber to the CE cabinet 4, 8, 12, 16, and 24 h after inoculation. Following the final transfer, lights were turned on inside the CE cabinet and set for a 12-h light/dark photocycle. When lesions became visible (approx. 2 days after inoculation), the number of lesions per mm2 of leaf surface was assessed; 4 days after inoculation, the number of necrotic and healthy leaves was counted. The above-mentioned experimental procedure was repeated with the dewchamber temperatures maintained at 12, 15, 20, and 25 °C. 2.5. Delayed dew periods Sixty pots of A. fatua seedlings (2 per pot ¼ 1 replicate) were grown to 3-leaf stage (Zadoks’ 13) and inoculated with a D. avenacea conidial suspension at a concentration of 5 104 conidia per ml. Seedlings were then subjected to the following dew-period treatments, all in the dark: 16-h dew period, 12 h in CE cabinet; 1 h in CE cabinet, 16-h dew period, 11 h in CE cabinet; 2 h in CE cabinet, 16-h dew period, 10 h in CE cabinet;
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4 h in CE cabinet, 16-h dew period, 8 h in CE cabinet; 6 h in CE cabinet, 16-h dew period, 6 h in CE cabinet; and 8 h in CE cabinet, 16-h dew period, 4 h in CE cabinet. Following the final transfer, lights were turned on inside the CE cabinet and set for a 12-h light/dark photocycle. When lesions were visible to the unaided eye (approx. 2 days after inoculation), the number of lesions per mm2 of leaf surface was assessed using a stereo dissecting microscope. Four days after inoculation, the number of necrotic and healthy leaves was counted. Treatment means were compared using the Tukey’s x procedure. 2.6. Repeated suboptimal dew periods Forty pots of A. fatua seedlings (2 per pot ¼ 1 replicate) were grown to 3-leaf stage (Zadoks’ 13) and inoculated with a D. avenacea conidial suspension at a concentration of 5 104 conidia per ml. Seedlings were then subjected to the following dew-period treatments, all in the dark: 6-h dew period, 22-h CE cabinet; 8-h dew period, 20-h CE cabinet; 6-h dew period, 12-h CE cabinet, 6-h dew period, 4-h CE cabinet; and 8-h dew period, 12-h CE cabinet, 8-h dew period. Humidity within the CE cabinet was maintained below 50% R.H. and the temperature was 25 °C. Following the final transfer, lights were turned on inside the CE cabinet set for a 12-h light/dark photocycle. When lesions were visible to the unaided eye (approx. 2 days after inoculation), the number of lesions per mm2 of leaf surface was assessed using a stereo dissecting microscope. Four days after inoculation, the number of necrotic and healthy leaves was counted.
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dew chamber at 20 °C under darkness for a period of 16 h and then placed in a CE cabinet at 25 °C. For five pots from each spore concentration, the number of lesions per mm2 of leaf area was assessed 4 days after inoculation. The remaining seedlings were allowed to grow for a further 6 days (a total of 10 days after inoculation) before the proportion of dead leaves was assessed and dry weights recorded. 2.9. Vegetative growth recovery Sixty pots of A. fatua seedlings (2 per pot ¼ 1 replicate) were grown to 3-leaf stage (Zadoks’ 13) and inoculated with a D. avenacea conidial suspension at a concentration of 5 104 conidia per ml. Ten of these plants were placed in a dark CE cabinet at 20 °C. The remaining plants were placed in a dew chamber at 25 °C with 10 plants being transferred to the CE cabinet 4, 8, 12, 16, and 24 h after inoculation. Following the final transfer, the lights in the CE cabinet were turned on and the plants subjected to a 12-h dark/light photocycle. After 4 days, the number of necrotic and healthy leaves on each plant was assessed. Leaves were classified as dead when greater than 80% of their tissue was necrotic. This assessment was carried out at weekly intervals for the following 3 weeks. 2.10. Statistical analysis All analyses, except where noted, were undertaken using the Genstat statistical analysis system (IACRRothamsted, Harpenden, Herts, England).
3. Results 2.7. Post-inoculation temperature 3.1. Plant age and susceptibility Two A. fatua seedlings were grown in 50, 10-cm-diam pots. When seedlings had matured to the 3-leaf stage (Zadoks’ 13) they were inoculated with a spore suspension at a concentration of 5 104 conidia per ml and placed in a dew chamber for 16-h at 15 °C under darkness. Ten seedlings were then placed in CE cabinets at 10, 15, 20, 25, and 30 °C. When lesions appeared, the number of lesions per mm2 was assessed for each treatment. The percentage of leaves with > 80% necrotic tissue was assessed 10 days after inoculation. 2.8. Inoculum concentration Sixty pots of A. fatua seedlings (2 per pot ¼ 1 replicate) were grown to the 3-leaf stage (Zadoks’ 13). Ten plants were inoculated with a spore suspension of 1 104 , 5 104 , 1 105 ; 2 105 , and 3 105 spores per ml of D. avenacea. Ten pots of seedlings were sprayed with water and Tween 20 only. All plants were placed in a
To compensate for the lower dry weight of young plants compared to older plants, data were log-transformed before analysis. Young seedlings (1- and 2-week postemergence) were less susceptible to D. avenacea than older seedlings. Disease severity was high when plants were from 3 to 6 weeks of age and was maximal when plants were inoculated approximately 4ð1=2Þ weeks after emergence (Fig. 1). Plants were highly susceptible when inoculated at 4 and 5 weeks postemergence. Over 50% of the total tissue was necrotic in plants inoculated at 3-, 4-, and 5-week postemergence. 3.2. Dew-period temperature It was not possible to examine dew period responses at temperatures greater than 30 °C as seedlings began to wilt and necrose independent of their inoculation state. A regression equation describing the interaction be-
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3.3. Delayed dew periods A delay of up to 8 h following inoculation did not reduce disease severity (P < 0:005; data not shown). 3.4. Repeated suboptimal dew periods
Fig. 1. Susceptibility of A. fatua seedlings of various ages to D. avenacea. Dry weights of seedlings inoculated with a conidial suspension of D. avenacea ð1 105 conidia=mlÞ and uninoculated controls were recorded 10 days after inoculation. Relative disease severity is the natural log þ0:1 of differences between the mean dry weights ðn ¼ 5Þ of uninoculated control plants and inoculated plants of varying ages. Data points ð Þ are the means of treatments. The regression y ¼ 0:1278x2 þ 1:183 3:341 (—) accounted for 92.3% of treatment variance.
tween dew period, temperature, and disease severity was generated using SigmaStat (Jandel, CA, USA). Dew period, the temperature during the dew period, and the interaction of these two variables had significant effects ðP < 0:05Þ on disease severity. The optimum temperature range for maximum disease severity was 20–25 °C, which was more pronounced as dew period increased (Fig. 2).
Fig. 2. The effect of post-inoculation dew period and temperature upon disease severity of A. fatua seedlings by D. avenacea. The regression z ¼ 1:2e ½ððx 22:4Þ=7:2Þ2 þ ððy 20:2Þ=7:9Þ2 was fitted to the data (r2 ¼ 0.88).
Due to its skewed nature, the data set was logtransformed. An unpaired t test was used to compare 6-h dew period with 6-h repeated dew-period treatments and 8-h dew period with 8-h repeated dew-period treatments. Following a 6-h dew period, an average of 0.29 lesions per mm2 of leaf tissue was recorded. Subjecting plants to a second 6-h dew period following a 12-h low humidity period (average 0.26 lesions per mm2 ) did not increase this disease severity. Following a dew period of 8-h, 0.26 lesions per mm2 formed. A second 8-h dew period following a 12-h delay resulted in an increase in disease severity (0.39 lesions per mm2 ; P < 0:05). 3.5. Post-inoculation temperature Lesions on plants incubated at higher temperatures following inoculation expanded more rapidly and coalesced resulting in large necrotic areas and finally entirely necrosed leaves. There was a linear relationship between post-inoculation temperature and disease development (Fig. 3). 3.6. Inoculum concentration Data were not distributed normally within treatments and therefore a log-transformation was undertaken before analysis. An exponential model was fitted to the
Fig. 3. The effect of temperature on disease severity following infection of A. fatua seedlings by D. avenacea. Data points ð Þ represent treatment means. The regression y ¼ 2:5 23 (—) accounted for 70.5% of treatment variance.
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4. Discussion
Fig. 4. The effect of D. avenacea inoculum concentration (conidia/ml) on the severity of disease on A. fatua seedlings. Data points ð Þ represent treatment means. The regression y ¼ 0:3 0:3ð0:9999813x Þ accounted for 83.7% of treatment variance.
data and accounted for 80% of the variance (Fig. 4). Disease severity was affected by the inoculum concentration applied to the seedlings (P < 0:05). Disease levels rose sharply as inoculum concentration was increased to 1 105 conidia per ml. Increased concentration beyond this point did not result in large increases in disease severity. 3.7. Vegetative growth recovery At 7, 14, and 21 days after inoculation, all treatments except the 4-h dew-period treatment resulted in greater necrosis than uninoculated controls (Fig. 5). Twentyeight days after inoculation there was no difference ðP < 0:05Þ in the percentage of dead leaves between treatments.
Fig. 5. Recovery in terms of vegetative growth of A. fatua following inoculation of seedlings with a conidial suspension of D. avenacea at a concentration of 1 105 conidia/ml.
An appreciation of the biology of a weed, its potential biological control agent, and their interaction within their environment is an essential prerequisite to the initiation of a biological weed control program (Wapshere, 1975). Where the biological control program intends the use of a mycoherbicide, a number of these biological parameters have often constituted constraints to the success of previous systems (Auld and Morin, 1995). Central to these parameters are the innate virulence of the pathogen and its ability to initiate disease under limiting moisture conditions. In Australia, wheat, the primary market for any chemical or biological herbicide intended for wild oat, is grown under dryland conditions and is dependent on natural rainfall. Rainfall is highest in winter in the wheat-growing area although somewhat erratic in occurrence. Wild oat control is required early in the cropping cycle, generally before the crop reaches tillering (Mullen et al., 2000). This is also the only time at which D. avenacea produces a significant impact on wild oat growth. Our data suggest that a window of opportunity exists for the optimum use of D. avenacea to control wild oat when seedlings are between 3 and 5 weeks postemergence (Fig. 1). No reliable data exist on dew-period lengths in the Australian wheat belt but they are thought to be typically under 12 h. Planting occurs in late autumn or early winter and therefore D. avenacea must be applied during early to mid-winter. Average minimum temperatures in southern NSW for this period are 2–8 °C (MetAccess (CSIRO, 1999) data for Wyalong [33°560 S; 147°160 E] 1989–1999 May, June, July). Because of low temperatures at the time of application of D. avenacea, it is important that the interaction between dew period dependence and temperature be studied. We found that the optimum temperature for maximum disease development was 20–25 °C. At 10 °C only minimal disease was recorded for dew periods less than 12 h (Fig. 2). In the absence of suitable periods of overnight dew, bioherbicide application strategies might also take advantage of free water on the leaf surface provided by rain. This water would be less predictable and the agent may need to withstand delays before the initiation of dew or repeated suboptimal moisture periods. D. avenacea remains viable and capable of causing severe disease on the leaf surface for up to 8 h following application. The severity of disease recorded following an 8-h dew period was increased by a second 8-h dew period 12 h after the first. However two 6-h dew periods did not improve the pathogens efficacy. Hence, D. avenacea is not totally reliant upon overnight dew to initiate disease but would also respond to rainfall or mist occurring at appropriate times. These events may occur during the day when temperature is more likely to
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favor infection. In practice, efficacy would depend on the occurrence of appropriate environmental conditions and would require immediate response from wheat farmers. The post-inoculation temperature also has an effect on disease severity. We assessed leaf necrosis as an index to disease severity in this case as it gives an indication of the rate of disease spread rather than the infection rate per se. It was noted that lesions spread and coalesced resulting in wholly necrotic leaves more rapidly at higher temperatures. This resulted in a linear relationship between post-infection temperature and disease severity (Fig. 3). Again this would result in the efficacy of a product based on this organism being subject to environmental fluctuation. In a similar study, following the infection of malesterile hybrids of maize in the United States of America, Bipolaris maydis (Nisikado and Miyake) Shoem., a pathogen closely related to D. avenacea was found to be ‘‘not an effective cool weather parasite’’ (Nelson and Tung, 1972). Disease development following infection by B. maydis was favored by dew periods of 12 h or more, dew-period temperatures of 24 °C or more, and high post-dew-period temperatures. The authors suggested that disease epidemics in cooler maize-producing regions in the USA failed to become established because of the temperature dependence of the pathogen. Similarly, because of its dependence on high temperatures for infection, D. avenacea may be more useful as a bioherbicide in warmer, northern wheat-growing regions in Australia. Given an ideal environment, the severity of disease approaches a maximum following the application of inoculum at a concentration of 1 105 conidia per ml (Fig. 4). At this and higher concentrations, total necrosis of all inoculated leaves often occurs. However, regrowth from enclosed meristems occurs quickly. Four weeks after inoculation, recovery is complete and there is no difference between inoculated and uninoculated control plants (Fig. 5). Were this to occur in the field, it may provide a competitive advantage for wheat seedlings over wild oat during a critical phase in the crop cycle, similar to that reported for competitive cultivars (Lemerle and Cousens, 1992; Seavers and Wright, 1999). Cultivars with higher growth rates in leaf and tiller production have an advantage over other grass species. Modeling has indicated that employment of competitive cultivars may lead to a reduction in the rate of build up of herbicide resistance by up to 50% (Gill, 1998) and therefore similar outcomes may result from the development and deployment of D. avenacea as a bioherbicide to control wild oat. Strategic application of D. avenacea as a biological control agent and the consequent weakening of A. fatua may have a place in integrated weed control, particularly given the spread of herbicide resistance. Two major
problems exist. First the dew period requirement for this pathogen is unrealistic at low temperature and tactical application to take advantage of unpredictable rainfall is not a practical option. Second, D. avenacea does not produce spores in submerged liquid culture as generally preferred for large-scale commercial production of bioherbicides (Auld and Morin, 1995). Generic formulation research underway in our laboratory may provide a solution to the first of these problems. The use of solid-state fermentation systems for the production of biological control agents is also an area of active research (Ooijkaas et al., 2000). Bioherbicides continue to suffer through direct comparison of their efficacy to chemical herbicides (Auld and Morin, 1995). An herbicide based on D. avenacea will not provide the efficacy in terms of mortality that a chemical herbicide may. However, it has the potential through infection and growth retardation of weed seedlings to provide a competitive advantage to crop seedlings. Additionally, it is a fairly specific pathogen (Hetherington and Auld, 2001), and pending technical solutions to water requirement and production constraints, remains a candidate as a biological control agent of wild oat.
Acknowledgment The authors wish to acknowledge the financial support of the Australian Centre for International Agricultural Research for providing funding to conduct the research.
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