Screening for non-host rotation crops of Colletotrichum acutatum for strawberry nurseries in California

Screening for non-host rotation crops of Colletotrichum acutatum for strawberry nurseries in California

Summary

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.

Objectives/Performance Targets

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.

Accomplishments/Milestones

Results and discussion for plant inoculation experiments

RESULTS
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 3.4×106 conidia/g were produced on flax whereas 8.9×108 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 1.3×106 conidia/g for clover to a high of 5.3×108 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 3.8×108 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).

DISCUSSION
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

RESULTS
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.

DISCUSSION
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.

Impacts and Contributions/Outcomes

Growers with a history of anthracnose should consider it possible that the fungus is perpetuating itself on rotation crops. The fungus is clearly 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 does not produce lesions under the conditions tested and therefore does not produce sporulating acervuli on living plant tissue. Yet, the fungus has the ability to lie dormant in dry plant debris for at least 1 month and then sporulate profusely upon exposure to moist conditions. There are significant differences, between the plant species tested and the degree to which the fungus sporulates. Though we are attempting to find non-host plants, this information on how the pathogen overwinters on several of the known rotation crops will allow a further vigilance in our efforts to know when infection occurs and from what source.

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