Final Report for GW08-015
Our results show the fungus can colonize nearly all plants tested thus far. This includes a number of the common strawberry nursery cover crops such as triticale, Sudangrass, Austrian winter pea and bell bean. We can isolate the fungus from both living and dead tissue of most of the species inoculated. We are also able to induce sporulation from surface sterilized plant debris. We have not confirmed sporulation from tissue on living plants.
Extracts from various plants did not inhibit the fungus. However, essential oils from several plants did show inhibitory properties.
Strawberry fruit production in California is dependent upon the annual production of clean transplants from certified nurseries in Northern California and Southern Oregon. Nurseries located in Oregon’s Klamath Co., and California’s Lassen, Siskiyou, Modoc, Shasta, Tehama, Glenn, San Joaquin, and Merced Cos. provide roughly 100% of the nursery stock required for California’s annual strawberry production system, and a significant portion of the stock used as the foundation for strawberries in other states and countries.
Strawberry nurseries rely heavily on chemical fumigants and fungicides for controlling pathogens and other pests. If the strawberry industry is to reduce its heavy dependence on these chemical control practices, effective alternatives for managing diseases and pests must be developed.
One method by which growers have attempted to avoid disease/pest problems is to adopt the practice of a multi-year crop rotation period between strawberry plantings. This can serve to break the disease cycle of various pathogens and pests by providing host-free periods. During these rotation years, nurserymen generally plant a cereal and/or legume crop.
Colletotrichum acutatum is representative of a large group of opportunistic fungal pathogens that affects a wide range of crops (Wharton and Diéguez-Uribeondo, 2004) such as strawberries (Smith and Black, 1990; Howard et al, 1992; Curry et al, 2002), blueberries (Smith et al 1996; Yoshida and Tsukiboshi, 2002), almond (Adaskaveg and Hartin, 1997; Forster and Adaskaveg, 1999), avocado (Freeman, 2000), peach (Adaskaveg and Hartin, 1997; Zaitlin et al, 2000), citrus (Zulfiqar et al, 1996; Timmer and Brown, 2000), mango (Arauz, 2000), and olive (Martin and Garcia-Figueres, 1999). Anthracnose is one of the most destructive diseases of strawberry in plant production nurseries and in fruit production fields. C. acutatum can infect all parts of strawberry plant causing crown collapse, leaf and petiole lesions, flower blight, and fruit rot. If infected plants are brought from nurseries to fruit production fields, crown rot generally will occur causing complete plant collapse. However, plant collapse seems to also require predisposing stress such as exposure to heat after digging the plants. Thus, plant collapse is generally not a problem in most years. Under conditions of rainfall, severe fruit rot occurs from inoculum brought into the field by transplants. Growers are dependent on fungicides for control of fruit rot. Crown rot has been reduced by simply washing soil away from the plants before planting or using fungicide dips of strobilurins (Daugovish and Gubler, 2006). However, complete control is dependent on clean plant production.
Colletotrichum spp. generally enter susceptible tissue of a primary host directly through the host cuticle by a penetration peg that emerges from a melanized appressorium. Appressorial development has been shown to be affected by numerous factors including temperature, light intensity, nutrient stress, and host surface characteristics (Emmett and Parbery 1975, Parbery 1981). Studies have shown that appressorial development is generally favored by cooler temperatures and severely impaired at temperatures of 35°C and above (Leandro et al., 2003a).
Experiments have shown that when C. acutatum is inoculated onto non-hosts such as pepper, tomato, garden bean, and eggplant, melanized appressoria are formed but do not appear to function (Horowitz et al., 2002). Some colonization of the tissue does occur but not via direct penetration from the appressoria. The fungus remains in a quiescent state in the epidermal cell layers without causing damage to the tissue. Its growth is restricted to the upper cellular layer beneath the cuticle where it is able to reach a balance with the plant, obtaining enough nutrients from the apoplast to survive but not taking so much that it harms the plant. The result is that the fungus is able to survive asymptomatically for extended periods (Horowitz et al., 2002). Whether or not these latent infections can be triggered to become active infections, and whether or not such infections will result in sporulation remains to be determined.
The fungus can survive in soil for at least nine months (Eastburn and Gubler, 1990). Though soil fumigation prior to planting the next strawberry nursery crop greatly reduced inoculum density, the fact that the pathogen is there during the fall-spring period indicates that various plants might become infected during this time. Moisture has also been shown to play a role in survival of the pathogen in soil (Eastburn and Gubler, 1992; Feil et al, 2003). Survival of C. acutatum has been found to be highest under cool, dry soil conditions and decreases with increases in both temperature and moisture.
At one point it was thought that crop rotation might be the answer to preventing inoculum carryover in strawberry plant production. Unlike Verticillium dahliae, C. acutatum lacks an identified resting structure. A several year host free period seemed like a sensible practice for reducing inoculum levels. However these rotation fields were noted to have been contaminated or infested by the presence of infected, volunteer strawberry plants (Gubler et al., 2006). Ideally, there would be no strawberry plants or plant material remaining after harvest. This may be an unrealistic goal.
To reduce inoculum density or prevent the possibility of pathogen increase in rotation fields in the vicinity of new plantations, it would be ideal to plant crops that suppress C. acutatum or at least are non-hosts to this pathogen.
It has been shown that some crops contain substances that have an antifungal effect. Mustard or pepper extracts suppress Verticillium dahliae (Bowers and Locke, 1998), Fusarium oxysporum (Bowers and Locke, 2000), and Phytophthora nicotianae (Bowers and Locke, 2004), and some Chinese herbs inhibit the growth of powdery mildew (Chu, et al., 2006). Cassia and clove treatments reduced soil populations of Fusarium oxysporum in controlled experiments (Bowers and Locke, 2000). Formulations of cassia extract and synthetic cinnamon oil reduced pathogen populations of Phytophthora nicotianeae (Bowers and Locke, 2004). Garlic extracts have been shown to inhibit growth of a wide range of soilborne fungal organisms (Wilson et al 1997; Sealy et al, 2007).
Palmarosa (Cymbopogon martini), thyme (Thymus zygis), cinnamon leaf (Cinnamomum zeylanicum), and clove buds (Eugenia caryophyllata) showed high levels of antifungal activity against Botrytis cinerea (Wilson et al 1997). Essential oils of cinnamon (Beg and Ahmad, 2002; Ranasinghe et al, 2002), clove (Beg and Ahmad, 2002; Ranasinghe et al, 2002), lemongrass (Paranagama et al, 2003), palmarosa (Velluti et al, 2004) thyme (Faleiro et al, 2003) and butterfly pea (Osborn et al, 1995) are known to be antifungal.
Compounds from several members of the family Rutaceae have been shown to have growth inhibitory components against Colletotrichum fragariae, Colletotrichum gloeosporioides, Colletotrichum acutatum, Phomopsis obscurans, Botrytis cinerea, and Fusarium oxysporum (Cantrell et al, 2005). In contrast, strawberry plants contain substances that stimulate sporulation of C. acutatum (Leandro et al., 2003).
1. Determine whether or not the commonly used strawberry nursery cover crops are a host for C. acutatum or if not a primary host, determine what role they may play in the survival of this fungus during the rotation period.
2. Screen crops that have possible inhibitory effects on the germination and growth of C. acutatum. Evaluate their potential as novel rotation crops.
Materials and methods for plant inoculation experiments
Inoculum production. Three to five isolates of Colletotrichum acutatum were used in these studies. Isolates were started by transferring mycelial plugs onto potato dextrose agar (PDA) plates and incubating at 25ºC for eight days. Cultures were then agitated, by scraping with tweezers, to induce sporulation. Five days later plates were flooded with DI water and a glass L-shaped rod was used to dislodge conidia. The suspension was filtered through three layers of cheesecloth to remove mycelia then vortexed for 1 min to break up spore clusters. The concentration was adjusted to 5x106 conidia/mL using spore counts from a haemocytometer. Several drops of Tween20 were added to break surface tension.
Plant production and maintenance for growth chamber (experiment 1) and greenhouse (experiment 2). Seeds of the plant species used in this study (Table 1) were purchased from Peaceful Valley Farm and Garden Supply (Grass Valley, CA) and Park’s Seeds (Greenwood, SC). Seeds were surface sterilized in 70% ethanol for 20 seconds then 0.5% sodium hypochlorite for two minutes and rinsed in sterile water. The seeds were germinated in petri dishes containing filter paper and sterile water, then seeded in 5-inch plastic pots with Sunshine Mix #1 (Sun Gro Horticulture, Vancouver BC). For experiment 1, seedlings were grown in growth chambers (UC Davis Controlled Environment Facility) at 25oC daytime and 18oC night at a constant 70% RH. Plants were watered with a 4-18-38 (Grow More Inc., Gardena CA) nutrient solution amended with calcium nitrate and magnesium sulfate and free of boron. There were 10 pots/species.
The trial was repeated in a greenhouse during the fall of 2008. The shift from growth chamber to greenhouse was made because we decided the precise environmental condition control, achieved in the chamber, was not necessary in order to complete our objectives. Cost and labor conditions were more favorable in the greenhouse. During experiment two, average temperature in the greenhouse was 18.8°C and the average relative humidity was 64.8%. Again 5-inch plastic pots were used this time with a Modified UC Mix for soil. Plants were maintained under drip irrigation with the same fertilizer described previously.
Experimental design for growth chamber (experiment 1) and greenhouse (experiment 2). This study used nine replicates per treatment (plant species). With replicates staggered over time in three chambers. Chambers were always set to 25oC daytime and 18oC night at a constant 70% RH and a 15 hr day. During each time interval, three replications of each treatment were grown, inoculated, and harvested from the chambers. In the greenhouse, the nine replications were arranged in a completely randomized design on a single table. For the sake of clarity, the chamber experiments will be referred to as “experiment 1a (infection), 1b (sporulation)” and the greenhouse experiments will be referred to as “experiments 2a, 2b” and so on.
Inoculations (experiments 1 and 2). Roughly nine plants of each species were inoculated with a 5x106 conidia/mL spore suspension by spraying the foliage to runoff using a 32oz spray bottle (The Bottle Crew, West Bloomfield MI). Plants were covered with clear plastic bags for 72 hr to maintain high RH. After inoculation, the plants remained in the growth chamber at 25oC daytime and 18oC night at a constant 70% RH for an additional 10 days.
Plant production and maintenance for field trials (experiments 3 and 4). Plants used in this study included: strawberry (Fragariae x ananassa Duch. cv. Albion), tomato (Solanum lycopersicum cv. Better Boy Hybrid), sesame (Sesamum indicum), red cowpea (Vigna spp.), bell bean (Vicia faba), hairy vetch (Vicia villosa), Austrian winter pea (Pisum arvense), sunn hemp (Crotolaria juncea), triticale (Triticum x Secale Juan.), Merced rye (Secale cereale), Sudan grass (Sorghum bicolor var. sudanense), wheat (Triticum urarta), mustard (Sinapsis), broccoli (Brassica oleracea cv. Packman Hybrid), rapeseed/canola (Brassica napus), and flax/linseed (Linum usitatissum). Six foot rows of seed were hand planted at 1 cm intervals into Yolo fine sandy loam soil at the Armstrong Experiment Farm (UC Davis). The field dimensions were 140 by 45ft. Rows were on 6ft centers, 130 ft long with 13 replicates per row. The spacing between replicates down the row was 4ft. Plants were sprinkler irrigated for about three hours two times per week. No fertilizer was added before or during the experiment.
Experimental design for Armstrong Farm field trials (experiments 3 and 4). Each treatment consisted of one plant species. There were 16 species and five replications. Each replicate consisted of the first few plants in the row. The replicates were arranged in a completely randomized design. The experiment was repeated by inoculating the last few plants in the row. The first inoculation took place on 10/13/08 at 6pm. The second inoculation took place on 10/29/08 at 5pm.
Inoculations (experiments 3 and 4). Each replicate was spray inoculated with a 5x106 conidia/mL suspension using a 2 gallon compressed air sprayer (R.E. Chapin Manufacturing Works, Batavia NY). Foliage was sprayed until runoff.
Plant production and maintenance for commercial field trial (experiment 5). Plants used in this study included strawberry (Fragariae x ananassa Duch. cv. ‘Albion’ Sierra Cascade Nursery Inc. Susanville CA), bell bean (Vicia faba, Peaceful Valley Farm and Garden Supply, Grass Valley CA), Austrian winter pea (Pisum arvense, Peaceful Valley Farm and Garden Supply, Grass Valley CA), Merced rye (Secale cereale, Peaceful Valley Farm and Garden Supply, Grass Valley CA), Sudan grass (Sorghum bicolor var. sudanense, Peaceful Valley Farm and Garden Supply, Grass Valley CA), mustard (Sinapsis, Peaceful Valley Farm and Garden Supply, Grass Valley CA) and rapeseed/canola (Brassica napus, Peaceful Valley Farm and Garden Supply, Grass Valley CA).
Three meter rows of seed were hand planted into a Cortina very gravelly sandy loam soil at a commercial nursery in Northern California on February 27, 2009. In 2008, this nursery site was cropped to strawberries, nearly all of which were infected by C. acutatum. The field was not harvested and so was left littered with infected strawberry plants. The spacing between replicates was three meters. Plants were overhead irrigated an average of two times per week. No fertilizer was added during the experiment.
To determine cleanliness of the Albion nursery stock, a total of 130 bare-root “white tag” plants were obtained for the trial. Seventy of those were planted at the commercial field site. Thirty of the remaining 60 plants were screened for latent infections using a method similar to that described previously (Mertely and Legard, 2004). The remaining 30 plants were planted in 12.7cm plastic pots and maintained in a greenhouse for symptom observation. C. acutatum was not detected from any of the plants so we assumed the other 70 were clean as well.
Experimental design for commercial field trial (experiment 5). Each treatment consisted of one plant species. There were seven species each with seven replicates. Each replicate consisted of a three meter strip of plants. The replicates were arranged in a completely randomized design on a single transect across the contaminated field.
Symptom development. Ten to thirteen days post inoculation the plants were inspected for lesion development. Suspect tissue was examined under a microscope for acervuli. When necessary, tissue was cleared by placing in a 12-well tissue culture tray and submersing for 24 hr in a 2:1 solution of glacial acetic acid and ethanol (2:1mL). Acid fuschin (0.05%) was used to stain fungal structures. Images were captured using a Leica DMLB compound microscope fitted with a Leica DFC480 camera using LAS software. Images were blended using AutoMontage software (Syncroscopy, Frederick MD).
Plant harvest. Once all plants had been inspected for symptoms, they were harvested by cutting at the soil line. The plants were then cut up into smaller pieces, leaves were saved, and stems were discarded. Plant material was treated by immersion in 70% ethanol for a brief dip and then 0.5% sodium hypochlorite for two minutes to kill any pathogen propagule on the leaf surface, then rinsed two times in sterile DI water. Each plant was kept in a separate bag. The effectiveness of surface treatment was verified by plating the rinse water on potato dextrose agar amended with tetracycline (PDA-tet). No colony forming units were detected from any of the rinse water. It is possible the sterilization technique was ineffective at killing appressoria still attached to the leaf surface. This was not tested. Thus, recovery of the pathogen following the alcohol/bleach treatment indicated it was present inside the tissue and/or as a durable propagule attached to the leaf surface.
Isolation from asymptomatic leaf tissue. Sterilized tissue was randomly selected from each of the bags and plated on (PDA-tet). Three plates for each of the plants were used with each plate containing 12 sections of tissue (36 sections total). Plates were incubated at 25ºC for five days. Fungal colonies that emerged from surface sterilized tissues were identified based on morphological characteristics.
Detection of C. acutatum from commercial nursery field cover crops (experiment 5). Entire plants were removed from the soil and rinsed free of soil under running water. Plants were then surface sterilized in 0.5% sodium hypochlorite for one minute. A previous study found more than one minute resulted in a reduction in detection frequency (Mertely and Legard, 2004). Sterilized tissue was randomly selected and plated on (PDA-tet). Five plates for each replicates were used with each plate containing 10 sections of tissue (50 sections total). Plates were incubated at 25ºC for 5 days. Fungal colonies that emerged from surface sterilized tissues were identified based on morphological characteristics.
Sporulation on surface sterilized tissue. The remainder of the plant material was placed in open brown paper lunch bags and placed in large crispers. The crispers were covered with three layers of cheesecloth to screen contaminants while allowing for desiccation to occur. The desiccation period was 30 days. At this time, 0.2 grams (dry weight) of debris was transferred to a 100 x 15mm Petri dish containing Whatman no. 1 filter paper covered by 1/8” plastic mesh (US Plastic Corporation, Lima OH). The contents of the plate, were sprayed with 8.5 mL of DI water and then incubated at 25ºC for seven days. There were three plates for each bag and four bags (reps) for each species. Each bag contained a single plant of that species. Following the incubation period, the contents of each plate was transferred to a 50mL polypropylene centrifuge tube (CLP VWR, West Chester PA) and flooded with 45mL of sterile DI water. Each tube was vortexed for one minute and the average number of conidia/mL was quantified using a haemocytometer.
Data analysis. Species differences in sporulation and isolation were assessed using one-way analysis of variance (ANOVA). For experiments 3 and 4, a two-way ANOVA with repeated measures was used to account for possible dependence introduced over time and possible differences in response over time due to species. Pairwise comparisons were made using either Tukey-Kramer or Fisher’s least significant difference test (LSD). If appropriate, data were transformed using the square root function (Rao, 1998). Model fit was assessed using graphical analysis of residuals and the Shapiro-Wilk test for normality. Statistical significance was expressed at the P = 0.05 threshold level. Analysis was performed using SAS Version 9.1 for Windows (SAS Institute, Cary, NC).
Materials and methods for extract/essential oil experiments
Plant Production and maintenance. Plants of the following species (Table 3) were grown in growth chambers at 25oC daytime and 18oC night at 70% RH.
Inoculum production. Cultures of C. acutatum isolates (Table 2) were started by transferring plugs of mycelia on potato dextrose agar (PDA) plates and incubating at 25°C for 8 days. Cultures were then agitated, by scraping with tweezers, to induce sporulation. Plates were then flooded with DI water 5 days later and a glass hockey stick was used to dislodge conidia. Suspension was filtered through 3 layers of cheesecloth to remove mycelia. The mixture was then vortexed for 1 min to break up spore masses. The concentration was adjusted to 5x105 conidia/mL using a haemocytometer.
Extract preparation. Concentrated leaf extract (1:9, wt/vol) was prepared according to the method described by (Leandro et al, 2003). Ten grams (fresh weight) of leaf tissue was ground in 100mL sterile deionized water (SDW) using a mortar and pestle. The resulting pulp was passed through four layers of cheesecloth into a flask. And the filtrate was centrifuged at 10,000 rpm for 5 minutes. The supernatant was vacuum filtered through Whatman No. 1 filter paper, and then filter sterilized through a 0.45-m-diameter Millipore membrane.
Paper disk agar diffusion assay. A modified version of the method described by (Kishore and Pande, 2007) was used. A 15-mm-diameter sterilized filter paper disk (Whatman No. 1) was placed in the center of a Petri dish containing PDA (1.5% agar). 50l of extract (1:9 wt/vol) or essential oil was loaded onto each of the disks. Paper disks loaded with sterile DI water were used as controls. After 30 minutes the plates were overlaid with a 10mL suspension of C. acutatum conidia in PDA (0.5% agar, 5x105 conidia/mL) collected from three isolates (Table 2). Plates were parafilmed. The plates were then left to incubate in crisper boxes at 25°C. Inhibition zones were measured after 72 and 144 hours. Parafilm was removed after 72 hours. There were 3 plates of each extract.
Effect of essential oil volatiles on growth of mycelium. A plastic vial cap was embedded in the center of a Petri dish containing potato dextrose agar (PDA). 250 µl of oil was added to the vial. 3 isolates of Colletotrichum acutatum were transferred onto the plate equally spaced surrounding the vial. The plates were parafilmed and incubated at 25°C. Colony diameters were measured at 72, 144, and 194 hours. Parafilm was removed after 144 hrs. The experiment consisted of 15 treatments (each oil is a treatment) plus a water control, with 3 sampling intervals and followed a completely randomized design with 5 replicates.
Effect of crude extract volatiles on growth of mycelium. Compartmentalized plates (double) were used. On one side was PDA with the fungus. On the other side was 3mL of the crude plant extract. Plates were parafilmed. Each extract was a treatment. Three isolates were used. There were 4 reps of each isolate with each treatment. Water was used as a control. Colony diameter was measured after 72 and 144 hours.
Statistical analysis methods. The results of different treatments and their effect on C. acutatum growth were assessed using one-way analysis of variance (ANOVA). Additionally, a two-way ANOVA with repeated measures was used to account for possible dependence introduced over time and possible differences in response over time due to treatment. Pairwise comparisons were made using either Tukey-Kramer or Fisher’s least significant difference test (LSD). If appropriate, data were transformed using the square root function (Rao, 1998). Model fit was assessed using graphical analysis of residuals and the Shapiro-Wilk test for normality. Statistical significance was expressed at the P = 0.05 threshold level. Analysis was performed using SAS Version 9.1 for Windows (SAS Institute, Cary, NC).
Tissue infection. The incidence of Colletotrichum acutatum in artificially inoculated asymptomatic plant material varied greatly among the species tested (F23,77 = 4.4, P = 0.0001). In experiment 1a, where plants were maintained at 25°C daytime 18°C night at a constant 70% RH, infection ranged from a low of 19.4% for millet to 79.4% for mustard (Figure 1A).
In experiment 2a, where temperatures ranged from 12.2-34.0°C and the RH fluctuated between 22.5-100% (Figure 2), similar results were observed (F23,109 = 6.3, P = 0.0001). In this experiment, incidence of infection ranged from a low of 8.3% for Sudan grass to a high of 90.3% for sesame (Figure 1B).
In experiments 3a and 4a, species was again statistically significant (F14,51 = 26.9, P = 0.0001). Incidence ranged from 0.5% for broccoli to 77.1% for sesame (Figure 3A). The experiment was repeated by inoculating different plants in each row, and species was a significant factor in the ANOVA (F14,49 = 15.7, P = 0.0001). Incidence ranged from 1.1% for canola up to 95.4% for sunn hemp (Figure 3B).
For experiments 3a and 4a over time, the interaction of species with time was statistically significant (F14, 51.5 = 10.6, P = 0.0001). Specifically, the pathogen was recovered from Austrian winter pea at a higher frequency than from wheat in experiment 3, while the reverse was true during experiment 4 (Figure 3). Additionally, Merced rye had a higher incidence of infection than mustard during experiment 3, while the reverse was true during experiment 4.
Sporulation on surface sterilized tissue. The quantity of C. acutatum conidia produced on artificially inoculated asymptomatic plant material varied greatly among the species tested (F23,127 = 6.6, P = 0.0001). In experiment 1b, where conditions were set at 25°C daytime 18°C night at a constant 70% RH, a low of 3.4x106 conidia/g were produced on flax whereas 8.9x108 conidia/g were produced on basil (Figure 4A).
In experiment 2b, where temperatures ranged from 12.2-34.0°C and the RH fluctuated between 22.5-100% (Figure 2), similar results were observed (F23,98 = 12.7, P = 0.0001). In this experiment, sporulation ranged from a low of 1.3x106 conidia/g for clover to a high of 5.3x108 conidia/g for strawberry (Figure 4B).
In experiment 4b, conducted under field conditions, species was a significant factor in the ANOVA (F14,49 = 8.8, P = 0.0001). No sporulation occurred on broccoli, hairy vetch, Merced rye, mustard, canola, triticale and wheat, yet 3.8x108 conidia/g was produced on strawberry (Figure 5B).
For experiments 3b and 4b, the interaction of species with time was statistically significant (F14, 55.2 = 7.6, P = 0.0001). Specifically, no sporulation occurred on red cowpea, bell bean, Austrian winter pea, sunn hemp and Sudan grass during experiment 3, while sporulation did occur on these plants during experiment 4 (Figure 3).
Our results showed that C. acutatum can asymptomatically colonize nearly all plant species tested. This includes a number of commonly utilized strawberry nursery cover crops. The fungus can be isolated from both living and dead tissue of most species inoculated under growth chamber, greenhouse and field conditions. Sporulation was induced from surface sterilized plant debris, suggesting internal colonization. Secondary conidiation was detected on many of the species tested indicating the pathogen has the ability to act as an epiphyte. This was not surprising considering a previous study had secondary conidiation occur on glass cover slips (Leandro et al, 2003b). Sporulation on living plants was not observed with the exception of a single marginal leaf lesion on butterfly pea (Figure 8) and stem lesions on a red cowpea plant (Figure 9) and a bell bean plant in the greenhouse. The red cowpea and bell bean plants that showed symptoms were leggy and had fallen over. Foliage from adjacent plants had grown over resulting in a “canopy effect”. The soil in the pots containing these plants was relatively dry. We think the reason symptoms were observed on these plants and not on any of the other replicates of these species, throughout any of the experiments, was because the symptomatic plants were stressed and moisture conditions were more favorable under the canopy. Additional lesions were detected on several other plant species. Leaves were cleared and stained for microscopy to look for sporulation from acervuli. None was observed
In experiments 1a and 1b pathogen recovery was relatively high for all species. Similarly, the number of plant species on which the fungus sporulated was relatively high. The same was true for experiments 2a and 2b. There were higher levels of sporulation from plants in experiment 1b than for experiment 2b. Although weather conditions were similar during experiments 1 and 2, greater extremes of both temperature and humidity during experiment 2 may have contributed to the variation observed between the two experiments (Figure 2).
Experiments 3 and 4 more realistically represented conditions the fungus and plant would experience in a commercial field. Plants were inoculated with a concentrated spore suspension but otherwise conditions would be similar. The weather data from these experiments varied greatly (Figures 6, 7) and the isolation/sporulation data reflects this (Figures 3, 5).
The optimum temperature for infection by Colletotrichum spp. is reported to be about 25°C (King et al, 1997, Wilson et al 1990, Dodd et al 1991, Nair and Corbin 1981, O’Connell et al 1993). One study found an optimum temperature range of 23 to 27.7°C for conidial germination within 12 h after inoculation (Leandro et al 2003a). The temperature the first 12 h following experiment 3 inoculations was significantly lower than this optimal range (Figure 6a). The average temperature during that period was 9°C. The first 12 h following experiment 4 inoculations were warmer, 14.9°C, but still well below optimal (Figure 6b). We observed higher infection levels during experiment 4 where, even though the average temperature for the entire period was lower, the average temperature during the first 12 h was 6°C higher than during experiment 3.
In addition to temperature, moisture conditions were more favorable during the second period as well. Free moisture is required for infection of fruit by C. acutatum (Wilson et al 1990). No precipitation occurred during experiment 3 (Figures 7a, 3, 5). During experiment 4, a significant amount of precipitation occurred and we observed higher levels of infection (Figures 7b, 3, 5).
It should be noted that in experiment three, sporulation was detected only on strawberry and sesame and only in one replicate in each case. Otherwise there was no sporulation during this experiment (Figure 5a).
Several studies suggest that plants may acquire resistance to Colletotrichum spp. as they mature. This was shown to be the case for C. coccodes on pepper (Hong and Hwang, 1998) and for C. truncatum on lentil (Chongo and Bernier, 2000). In our experiment we observed higher levels of infection during experiment 4. We believe this was due to factors other than plant age.
One possible explanation for the increased infection levels during experiment 4 is the plants could have become infected following the experiment 3 inoculations in which case the fungus would have had time to grow and further infect the tissue. We do not believe this is a problem for several reasons, 1) there was approximately 1.2m of buffer plants between the first and last plants in the row, 2) we took care not to overspray our inoculum, 3) apart from some possible secondary conidiation, the fungus does not sporulate on living tissue of the plants tested which could have provided an additional source of inoculum to be splash dispersed onto adjacent plants, and 4) even if some infection occurred, it should not have differentially affected the plant species being tested, as all were treated in the same way.
Regardless of what caused the differences in infection/sporulation observed during the various experiments, the overall trend observed for the treatment effect remained consistent. Sesame and red cowpea tended to have a higher incidence of the fungus in inoculated tissue whereas rapeseed and Sudan grass tended to have the lowest (Figures 1, 3). Higher levels of sporulation were consistently detected on strawberry, red cowpea and bell bean while low levels tended to be produced on alfalfa and tomato (Figures 4,5).
It has previously been demonstrated that plant pathogenic Colletotrichum species are able to asymptomatically colonize plants and express nonpathogenic lifestyles (Redman et al, 2001; Freeman et al, 2001; Horowitz et al, 2002). Redman et al showed several pathogenic Colletotrichum spp. expressed either mutualistic or commensal lifestyles in non host plants. C. acutatum was found to be a commensal on several cultivars of watermelon and an endophyte on others (Redman et al, 2001). Cucumber, squash, tomato, pepper, bean, and corn were also reported to be non-hosts of the fungus (Redman et al, 2001). Their criterion for designating “non-host” status was when the pathogen could not be detected in the tissue at all, not even asymptomatic colonization.
In our studies we were able to isolate the fungus from asymptomatic leaves of tomato and corn. The cultivars used in our experiments were different from those used by Redman et al. The tomato cultivars used by Redman et al. were ‘Seattle Best’, ‘Roma’ and ‘Big Beef’. ‘Better Boy’ hybrid was the tomato cultivar used in our study. The corn cultivars used by Redman et al. were ‘Jubilee’ and ‘Kandy Korn’. ‘True Gold Sweet’ was the corn cultivar used in our study. Thus it seems that cultivar might play an important role in susceptibility.
The inoculation technique used in our experiments was different than the technique described by Redman et al. Our inoculation technique involved spraying above ground plant parts with a spore suspension. Redman et al’s technique involved immersing the roots and lower stem in a suspension. The Colletotrichum acutatum isolate used in their experiments was pathogenic on the two strawberry cultivars tested, ‘Sweet Charlie’ and ‘Rosa Linda’. Their criterion for labeling a “pathogenic” interaction was plant mortality. C. acutatum is well known to produce symptoms on nearly all strawberry plant parts. Infection of these parts does not always result in plant mortality.
The disease cycle of Colletotrichum acutatum is likely similar to that of the prokaryotic angular leaf spot pathogen, Xanthamonas fragariae, in strawberry nurseries. It is not uncommon for there to be crop debris in and around nursery fields. Previous work has shown the fungus can colonize strawberry leaves and petioles without causing symptoms (Leandro et al 2001; Horowitz et al, 2002; Mertely and Legard, 2004), and our work has shown that once strawberry leaves senesce and are subjected to moisture, the fungus sporulates. Other studies have shown the fungus can be triggered to sporulate from asymptomatic debris by artificially inducing senescence with the use of fungicides (Cerkauskas, 1988; Sinclair, 1991) or freezing temperatures (Mertely and Legard, 2004).
Prior to digging, it is common practice for nurserymen to mow their strawberry fields. Leaves and petioles from a contaminated field can blow to adjacent fields where once dried or frozen or treated with herbicide, the fungus will sporulate. Conidia may be deposited on whatever plants are in the vicinity. These plants can be colonized asymptomatically. Inoculum will build up as leaves senesce and are exposed to moisture. At the end of the cover crop period, it is common for nurserymen to mow the cover crop prior to incorporating the material into the soil. At this time inoculum can be transferred to adjacent fields or fence lines in the form of asymptomatic infections on the cover crop.
Growers with a history of anthracnose should consider it possible that the fungus is perpetuating itself in rotation fields. C. acutatum is able to asymptomatically colonize a variety of rotation crops. It is also able to produce secondary conidia on the leaves of the plants tested. The fungus did not produce lesions under the conditions tested and therefore did not produce sporulating acervuli on living plant tissue. Yet, the fungus has the ability to lie dormant in dry plant debris for at least one month and then sporulate upon exposure to moist conditions. There were significant differences, between the plant species tested and the degree to which the fungus sporulates on infected tissue.
Our growth chamber and greenhouse experiments were conducted under artificial conditions that favored the fungus. Even our field trials involved inoculating crops with high concentrations of spores and then maintaining them under overhead irrigation. Thus it is possible that our data overstate the capacity of C. acutatum to colonize the rotation crops that were tested. We are currently conducting field trials with commercial nurseries which should indicate the likeliness that natural inoculum can lead to infection of rotation crops.
Results and discussion for extract/essential oil experiments
Paper disk agar diffusion assay. The diameter of the inhibition zone varied greatly among the essential oil treatments tested at both 72 (F13,42 = 83.2, P = 0.0001) and 144 hours (F13,42= 27.8, P = 0.0001). After 72 hours, inhibition zones ranged from 0mm for Roman chamomile oil up to 85mm for thyme and peppermint oils (Figure 10a). After 144 hours, inhibition zones ranged from 0mm for Roman chamomile, garlic oil and tea tree oil up to 85mm for thyme oil (Figure 10b). Over time, the interaction of treatment with time was statistically significant for this assay (F13,42 = 12.32, P = 0.0001). Specifically, inhibition occurred on tea tree and garlic oil plates after 72 hours yet by 144 hours the fungus had overgrown these areas. None of the plant extract treatments tested inhibited growth of the fungus.
Effect of essential oil volatiles on growth of mycelium. The peppermint, cassia, tea tree, lemongrass, thyme and Roman chamomile treatments completely inhibited the fungus for all three isolates at all three time intervals and therefore they were not included in the statistical analyses. Colony diameter varied greatly among the other treatments tested at 72 (F8,36 = 24.9, P = 0.0001), 144 (F8,36 = 23.5, P = 0.0001), and 194 hours (F8,36 = 15.7, P = 0.0001). After 72 hours, colony diameter ranged from 0mm for basil, citronella and spearmint to 14mm for the sterile water control (Figure 11a). After 144 hours, colony diameter ranged from 0mm for basil, citronella and spearmint to 30mm for the sterile water control (Figure 11b). After 194 hours, colony diameter ranged from 0mm for citronella and spearmint to 38mm for the sterile water control (Figure 11c). Over time, the interaction of treatment with time was statistically significant for this assay (F16,324 = 4.8, P = 0.0001). Specifically, the basil oil treatment completely inhibited the fungus after 72 and 144 hours yet by 194 hours some growth had occurred.
Results were the same for all three isolates so only the data for one of these (Ca-50-05) was reported.
Effect of crude extract volatiles on growth of mycelium. There was no distinguishable difference between any of the crude plant extract treatments and the water controls at either time interval. Plant extract treatments neither inhibited nor promoted growth of the fungus.
A variety of essential oils effectively inhibited growth of C. acutatum. Contact was not required for inhibition to occur. Volatiles given off by the essential oils of peppermint, cassia, tea tree, lemongrass, thyme, Roman chamomile, spearmint, citronella and basil were all nearly 100% effective at inhibiting growth of the fungus. The essential oil of thyme was the all around most effective treatment based on results from both assays.
These experiments involved highly concentrated essential oils. At these rates application of these products in agricultural settings would be prohibitively expensive. In addition, these concentrations may result in phytotoxicity. Further work is required in order to determine if these oils are still effective at inhibiting growth of the fungus at dilute, economically feasible rates.
Crude plant extracts did not inhibit growth of C. acutatum during any of our experiments.
Education and Outreach
1. 2008 - Jertberg, J.R. and Gubler, W.D. Survival of Colletotrichum acutatum on common
strawberry nursery cover crops. American Phytopathological Society – Annual Pacific Division Meeting- Oral Presentation.
2. 2009 - Jertberg, Joseph Robert. Biology and control of Colletotrichum acutatum on
strawberry. M.S. Thesis. UC Davis Department of Plant Pathology.
3. 2009 - Jertberg, J.R. and Gubler, W.D. Survival of Colletotrichum acutatum on common
strawberry nursery cover crops. Plant Disease (In Preparation)
4. 2009 - Jertberg, J.R. and Gubler, W.D. Effect of crude plant extracts and essential oils on growth of Colletotrichum acutatum. Plant Health Progress (In Preparation)