Progress report for GW17-032
Fusarium oxysporum f. sp. fragariae (FOF), causal agent of Fusarium wilt of strawberry, was first detected in California in 2006, in fields where pre-plant fumigation with methyl bromide and chloropicrin was no longer used. The disease is now found in all major strawberry production areas and methyl bromide, a key ingredient in fumigation mixtures, is expected to be unavailable by 2017. Only partial suppression of Fusarium wilt can be achieved by fumigation with chloropicrin alone. Some strawberry cultivars are resistant to Fusarium wilt, but most commonly grown cultivars are susceptible. Furthermore, preliminary studies indicate that the Fusarium wilt pathogen can colonize resistant strawberry plants and produce structures that survive in soil (=inoculum). Consequently, growing a resistant strawberry crop may contribute to increases in soil inoculum, which may eventually exceed levels resistant cultivars can tolerate. Therefore, there is an urgent need to identify rotation crops that will not support growth of the pathogen.
To address this question, the biomass production of FOF on crops that are commonly grown in rotation with strawberries and on FOF resistant strawberry cultivars were measured using culture dependent methods. Results showed raspberry, a common rotation crop with strawberry, can maintain or increase populations of this pathogen in soil. Resistant strawberry cultivars, while asymptomatic, can also become extensively colonized by this pathogen. A survey to investigate producers' ability to rotate with specific crops showed crop rotation to be a common disease management practice in the Watsonville/Salinas production region. Survey participants indicated that more control over land leases or access, and training/experience with growing crops other than strawberry, would improve their ability to use rotation as a disease prevention strategy.
Results of this research were presented at one field day and two grower meetings. In sum, results were communicated to over 300 growers, pest control advisors, and agricultural consultants/researchers. An information brochure was distributed at these events, which succinctly communicated the key findings. Multiple agricultural consultants and extension professionals indicated they now recommend against rotations with raspberry.
1. Research: Identification of crops that do not serve as reproductive hosts for FOF: Identify the relative potential of common rotation crops (lettuce, spinach, broccoli, cilantro, raspberry, wheat) to serve as reproductive hosts of Fusarium oxysporum f sp. fragariae (FOF).
2. Research: Inoculum contribution by resistant strawberry cultivars: Identify the extent to which cultivars of strawberry (Fragaria x ananassa) that are resistant to FOF can serve as reproductive hosts of this pathogen.
3. Extension: Survey on Crop Rotation for Disease Suppression: Survey growers, pest control advisors, and other industry representatives to identify barriers to adoption of crop rotation as a disease management strategy for FOF. This survey will be conducted in the winter and spring of 2017
4. Extension: Dissemination of results: Results of our research on crop rotation will be disseminated through the following types of publications: peer-reviewed, social media, websites, California Agriculture Journal, and informational pamphlets. All University of California Cooperative Extension (UCCE) professionals and notable pest control advisors will be contacted and informed of research results and their application. Results will be presented at three industry meetings and a workshop.
- Research: Identification of rotation crops that do not serve as reproductive hosts to Fusarium oxysporum f. sp. fragariae (cause of Fusarium wilt of strawberry; FOF)
Cultivars of rotation crops are commonly grown in the main strawberry growing regions of California, and were identified in collaboration with growers and UCCE agent Steve Koike:
- Lettuce (Lactuca sativa) cv. Green Towers OP
- Spinach (Spinacia oleracea) cv. Whale F1
- Broccoli (Brassica oleracea) cv. Marathon F1
- Cilantro (Coriandrum sativum) cv. Leisure
- Raspberry (Rubus idaeus) cv. Heritage
- Bread wheat (Triticum aestivum): cv. Summit
- Strawberry (Fragaria x ananassa): cv. San Andreas (resistant to FOF)
- Strawberry (Fragaria x ananassa): cv. Albion (susceptible to FOF)
Population growth on living crop tissue:
Dormant raspberry canes, strawberry crowns, and seeds of other crops will be planted seven days prior to the start of the experiment in #1 Sunshine mix in a growth chamber with a 16 hour light (25°C) and 8 hour dark (18°C) photoperiod. Upon germination, seedlings will be transplanted into a 1:3 mixture of sand infested with a hygromycin resistant, virulent strain of FOF (GL1080hyg+) and #1 Sunshine mix. Final inoculum concentration of the mixture will be assayed at the start of the experiment, and should be ~250,000 CFUs/g. Plants will be grown for 6 weeks under the previously described growth chamber conditions.
After the growth period (6 weeks), plants will be gently removed from the soil matrix. Soil from each pot will be passed through a 4mm sieve to capture/remove all roots, bagged, and stored at 4C until further analysis. Roots will be washed twice in 1% sodium hexametaphosphate to remove adhering soil particles and patted dry with paper towels. For raspberry and strawberry: the new roots that grew during this experiment will be separated from the older roots that were already formed in the dormant plants. Differentiation of old and new roots will be achieved through color and architectural differences: new roots are white, whereas the older roots have a brown external appearance. Branching and feeder roots will be removed from the central taproot in all crops except for wheat, which does not produce such a structure. The cortex will be removed from the stele for all other crops for separate analysis. Above-ground stem/leaf tissue will be isolated for each crop from tissue that is 1-5cm above the soil line. The compartments that will be assayed for each crop are shown in Table 1.
Fresh weight for each compartment of each biological replicate of each crop will be recorded, liquified with 100mL of sterile 0.1% water agar, serially diluted, and plated on 4 plates of Komada’s medium amended with 100mg/L of hygromycin B (1). Colonies will be counted 7 days later, and CFU/g of each tissue compartment calculated to provide a measure of pathogen biomass.
This experiment will be repeated three times, and each experiment will contain four biological replicates.
Soil inoculum survival:
The previous experiments assessed pathogen growth on living crop tissues. Additional population growth may occur in the rhizosphere or on plant tissues after tillage during decomposition. To assess the full contribution of FOF inoculum to the soil by rotation crops, we conducted a series of experiments to assay soil over a period of six months after incorporation of crop residue into soil.
Seeds of six rotation crops and dormant strawberry crowns were planted 14 days prior to the start of the experiment. Suckers from 1-year old raspberry canes were isolated at the time of planting. Crop types and cultivars were the same as those used in the previous experiment. 10 liters of sand infested with GL1080hyg+ was mixed with 15 liters of sterile sand and 63 liters of soil from a strawberry field near Watsonville, CA that had been passed through a 4mm mesh sieve. Potting soil was gently washed from the roots of germinated seedlings, and four biological replicates per crop type were transplanted into 2L pots containing the aforementioned soil mixture. An additional four pots were left un-planted as a “fallow” treatment. Pots were transferred to a controlled environment facility with a 12 hour photoperiod and temperatures of 28 C high / 20 C low. Plants were checked daily for 6 weeks and all pots (including fallow treatment) irrigated with a dilute N, P, and K solution when soil was dry.
After this period, fresh weight of above-ground plant biomass was recorded, then plant biomass cut into <1cm fragments, mixed evenly with the soil, and returned to the pot. For the fallow treatment (no plants), soil was removed, mixed, and returned to the pot. Soil containing plant debris was then returned to the growth chamber and irrigated weekly with 200 ml of charcoal-filtered water.
Soil was sampled from the bulk substrate (6 subsamples) at the beginning of the experiment, at the time of plant debris incorporation, and 12, 24, and 48 weeks post plant incorporation. The abundance of GL1080hyg+ in the soil was quantified by diluting soil with 1% sodium hexametaphosphate solution (to improve aggregate dispersal) and plating onto Komada’s medium with 100mg/L hygromycin. Two technical replicates were conducted per biological replicate per timepoint. The average of the technical replicates values was used for further analysis.
2. Research: Inoculum contribution by resistant and susceptible strawberry cultivars
Experiments will be carried out using the same protocol as described for assessing FOF population growth on living crop tissues in objective 1, except the tests will be done on five commonly grown, resistant cultivars of strawberry. The susceptible cultivar, ‘Albion’, will be used as a positive control. The cultivars to be tested are:
- Strawberry: cv. Albion (susceptible control)
- Strawberry: cv. Monterey (susceptible control)
- Strawberry: cv. San Andreas (resistant)
- Strawberry: cv. Ventana (resistant)
- Strawberry: cv. Fronteras (resistant)
- Strawberry: cv. Portola (resistant)
- Research/Extension: Survey on Crop Rotation for Disease Suppression
Our survey will be created in coordination with UCCE collaborators to identify the barriers of adoption of crop rotation. Questions will have categorical responses and optional comment boxes to facilitate the creation of a survey that can be completed in less than five minutes. Utilizing our existing network with pest control advisors and growers, we will disseminate this survey at annual grower meetings, over the telephone, via email, and by mail. The survey will be anonymous and approved through the UC Davis Institutional Review Board. Participants will be offered both Spanish and English versions of the survey. Responses will be compiled and analyzed for trends in commonly perceived advantages and disadvantages to crop rotation for disease prevention.
Komada, H. 1975. Development of a selective media for quantitative identification of Fusarium oxysporum from soil. Review of Plant Protection Research 8:114-115.
Suga H., Hirayama Y., Morishima M., Suzuki T., Kageyama K. and Hyakumachi M. 2013. Development of PCR primers to identify Fusarium oxysporum f. sp. fragariae. Plant Disease. 97:619-625.
Results from assays of living crop tissues showed resistant strawberry cultivars and raspberry consistently served as the best reproductive host for Fusarium oxysporum f. sp. fragariae (FOF), despite the complete lack of symptoms. The pathogen reproduced most extensively in the crown cortex, and >2mm diameter transplant roots of resistant cultivars, but was not seen at significant levels in feeder roots or crown stele tissues. For example, the crown cortex of cv. 'San Andreas' contained 16,400 CFUs/gram on average, and FOF was detected in the cortical tissue of 88% of plants. In contrast, average CFUs/gram on all other crops' taproot cortex were less than 200, and the pathogen was observed on cortical tissue of 0-22% of plants. The results were similar with the other resistant strawberry cultivars (Fronteras, Portola, and Ventana).
Crown stele tissues of susceptible cultivars (cv. Albion and cv. Monterey) were the most densely colonized by this pathogen, and contained 100,000-1,000,000 CFUs/gram. Cortex and stele populations were compared within cultivars by a paired t-test and Bonferroni correction applied to the resulting p-values. The susceptible cultivar, Albion, had significantly higher stele populations of F. oxysporum f. sp. fragariae compared to cortical populations (P = 0.006), but cv. Monterey did not (P = 0.10). In contrast, all resistant cultivars had significantly lower populations in the stele than in the cortex (P < 0.005). Growers with infested fields may consider removal of symptomatic plants to prevent these populations from being returned to the soil.
The soil inoculum survival experiments showed raspberry could significantly increase soil populations of F. oxysporum f. sp. fragariae after tillage. By 24-weeks post incorporation, a statistically significant increase in soilborne FOF population (roughly double the population present at time of tillage) was observed after growth of raspberry plants. Both susceptible and resistant strawberry cultivars showed no significant change in population between time of tillage and 24-weeks post incorporation. Net decreases in the amount of FOF was observed in un-planted pots and after all vegetable/wheat crop treatments. These results show that raspberry is a risky rotation crop in fields where F. oxysporum f. sp. fragariae is present. If raspberry is grown in such fields, the population of this pathogen can be expected to increase.
These data suggest that broccoli, lettuce, spinach, cilantro, or wheat would serve as weak reproductive hosts for FOF. Rotating with these crops may lead to decreases in soil inoculum as the amount of this pathogen in the soil decreases with attrition. In contrast, FOF-resistant strawberry cultivars and raspberry may allow greater reproduction by FOF and lead to persistence of this pathogen over time. There is also a risk of pathogen mutation to overcome resistance if these cultivars are planted year-after-year in infested fields. For these reasons, resistant cultivars should not be deployed as a stand-alone disease management strategy, but instead be integrated with other tools, such as crop rotation, for controlling this disease.
The survey on crop rotation practices was distributed at the 2018 Annual Strawberry Production Research meeting in Watsonville, CA. Fourteen meeting attendees responded to the survey. Respondents were evenly split between practicing organic, conventional, or both organic and conventional production practices. 85% of respondents regarded crop rotation as important for disease management in their production practices. Only 25% of respondents indicating that none of their acreage had been rotated to other crops in the past year, and all fields two years ago were rotated to strawberry from other crops. More control over land access/leases was most frequently cited as something that would improve ability to rotate strawberries with other crops (42% of respondents). 28% of respondents indicated that experience growing crops other than strawberry would also improve their ability to rotate. In sum, crop rotation is commonly practiced in the Watsonville/Salinas strawberry production region of California, but opportunities exist to improve growers' ability to use this strategy.
Educational & Outreach Activities
Summer 2017: I presented research on the benefits of crop rotation for control of Fusarium wilt of strawberry, and the potential risks of relying solely on planting resistant strawberry cultivars for disease control, at the annual "Farming without Fumigants" field day. This field day is put on by the California Strawberry Commission to raise awareness of disease management practices that could reduce reliance upon fumigation for disease control. Consultations about my research were conducted with participants.
Winter 2018: This research was presented at the 2018 Annual Strawberry Production Research Meeting in Watsonville, CA on February 1. Over 200 people were in attendance, including both growers and agricultural consultants/researchers. A handout describing the life cycle of F.o. fragariae and a list of good crops for rotations was distributed at this event.
Sprint 2018: This research was also presented at the 2018 Fumigation and Alternatives Strawberry grower's meeting in Ventura, CA on May 18. Fifty-eight growers, pest control advisers, and agricultural consultants/researchers were in attendance. A handout describing the life cycle of F.o. fragariae and a list of good crops for rotations was distributed at this event.
Crop rotation for control of Fusarium wilt diseases does not work if the crop can become extensively colonized by the population. We identified five common rotation crops that will not support increases in pathogen inoculum, and two that can maintain or increase the inoculum of this pathogen in soil. These data are critical for growers' to effectively practice crop rotation. These data will lead to improved adoption of this practice, and by preventing high-risk rotations can improve yield.
All disease management practices must occur within a broader production scheme, which can limit the ability to adopt specific practices. We identified key barriers to adoption of crop rotation by strawberry growers in the Watsonville/Salinas production regions, specifically: having more control over land leases or access to land, and more experience growing other crops. This key insight can be used to inform future projects to improve access to crop rotation as a disease management strategy by strawberry growers in California.
Providing alternatives to chemical-based management of plant diseases remains an important research priority. Our work shows that crop rotation can be used to manage Fusarium wilt of strawberry, but economic and knowledge barriers may still limit adoption of this practice. It is extremely difficult to design production practices that can directly replace fumigation for strawberry growers, because few biologically-based options exist with a comparable time and financial requirement. Therefore, other changes in production practices would need to occur to enable a shift from fumigants. For example, growers may need to diversify their production operations, and grow other crops in addition to strawberries to have the control over rotation that is necessary to effectively utilize this technique.