Pathology

A Comprehensive Approach to Management of Wilt Diseases Caused by Fusarium oxysporum and dahliae

Principle Investigator Dr. Thomas R. Gordon University of California Department of One Shields Avenue Davis, CA 95616 (530) 754-9893 [email protected]

Co-Principal Investigators Steven T. Koike, UC Cooperative Extension Oleg Daugovish, UC Cooperative Extension

Cooperators Douglas V. Shaw and Kirk D. Larson Plant Sciences Department U.C. Davis

Summary Our research in 2011 was directed toward the study of vascular wilt diseases caused by the soilborne pathogens, Verticillium dahliae and Fusarium oxysporum f. sp. fragariae. A central focus was on development and characterization of genetic resistance, which will make an increasingly important contribution toward management of wilt diseases in the future. Selecting for resistance to over many years has significantly increased levels of resistance to this disease, and in 2011, 63% of 61 breeding lines tested had resistance scores of 4.5 or higher (on a 1 to 5 scale, with 5 being a disease-free plant). We conducted a second year of tests in naturally infested soil to confirm the efficacy of resistance to Verticillium wilt. The results showed that ranking of cultivars based on susceptibility was essentially the same when infection occurred from exposure to inoculum in soil as when plants were root-dip inoculated. This indicates that genotypes identified as resistant by the screening procedure used in the University of California (UC) breeding program should be resistant under field conditions as well. In 2011, we continued development and implementation of a procedure to screen for resistance to Fusarium wilt. This included a comparison of inoculations with a single isolate and a mix of five isolates, and also a comparison of two different inoculum levels. Although disease was somewhat more severe in plants inoculated with the higher dose, the ranking of genotypes was similar. There was not a significant difference in results obtained with the single isolate or the mix.

9 2011 - 2012 RESEARCH PROJECTS As in past years, ‘Ventana’ and ‘San Andreas’ proved to be resistant to Fusarium wilt, whereas ‘Camarosa’ and ‘Albion’ were highly susceptible. Resistance scores for 26 breeding lines ranged from 1.0 to 5.0, with a mean of 3.3. Because grower observations suggested that soil acidification might render plants more prone to Fusarium wilt, we conducted experiments to test for an effect of pH on development of disease. Our results indicate that soil pH has at most only a weak effect on disease development, at least under controlled conditions. We continued to monitor the occurrence of Fusarium wilt and dieback caused by Macrophomina phaseolina, within California. Although Fusarium wilt has thus far been a serious problem only in Ventura County, recent finds indicate it may now also be established in the Santa Maria and Watsonville areas. Macrophomina has been confirmed to occur in all of the major strawberry production regions in the state.

Introduction Historically, Verticillium wilt, caused by the pathogenic soilborne , Verticillium dahliae, has been a major constraint on strawberry production in California. This disease remains a serious problem for organic strawberry growers and is of increasing concern for conventional producers where flat fumigation with methyl bromide and chloropicrin is no longer an option. When a plant suffering from Verticillium wilt dies, the pathogen produces large numbers of melanized, multi-cellular survival structures known as microsclerotia, which are incorporated into soil with crop residue. Microsclerotia can survive for one or more years and infect the roots of a susceptible crop subsequently grown at the same location. Even in fields where pre-plant fumigation has been applied for many years, the fungus can still be detected. Once fumigation is discontinued or ceases to be fully effective, populations of V. dahliae can be expected to increase to damaging levels. The risk is greatest where strawberries are grown in rotation with crops such as potato (high elevation nurseries) and lettuce (fruit production fields), both of which are susceptible to the same strain ofV. dahliae that causes disease in strawberries.

Where pre-plant fumigation cannot eliminate the risk of disease caused by V. dahliae, the extent of damage from Verticillium wilt will likely be determined primarily by the susceptibility of the crop. In other words, it will be necessary to develop strawberry cultivars that are resistant to the disease. In many crops, resistance has been achieved through breeding to combine a single gene for resistance with the characteristics required for a commercial cultivar. However, the efficacy of single gene resistance is often short-lived, as pathogen strains capable of overcoming it can quickly increase in abundance. The alternative is to develop disease resistance based on multiple genes. A longer timeframe is needed to achieve this type of resistance but it is likely to be much more durable. This is the approach being taken by the UC strawberry breeding program.

Since 1994, all strawberry genotypes used as parents have been tested for susceptibility to Verticillium wilt, as part of a multiple trait selection strategy. By using resistance to Verticllium wilt as a selection criterion, resistance scores for the parents used in the UC breeding population have increased by over 60%, and the percentage of genotypes that are at least moderately resistant has increased from 35% in the original germplasm to 78.5% in genotypes used as parents for recent crosses (Shaw et al., 2010a). As a result, breeding lines advanced to cultivar status are typically more resistant than cultivars released in earlier years. This report describes the results of the annual screening conducted in spring of 2011.

One objective of our research has been to verify that strawberry genotypes identified as resistant to Verticillium wilt based on the root-dip inoculation test used in the screening program will manifest resistance when exposed to V. dahliae under field conditions. For this purpose, we have developed naturally infested soils in which to grow plants that differ in susceptibility to Verticillium wilt based on a root-dip assay. The first tests were conducted during the 2009-10 season and the results showed that disease severity resulting from root dip inoculations and from exposure to soilborne inoculum were highly correlated. This report describes results from the second year of the study.

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Plant collapse caused by Fusarium oxysporum has become a serious problem in southern California production areas (Koike et al., 2009). It is not known when this pathogenic strain was introduced to California but it seems likely that its recent emergence is due to changing production practices. In particular, the combination of less effective materials (i.e., fumigants not including methyl bromide) and less complete treatment of soil (application to beds rather flat fumigation of an entire field) has allowed the Fusarium wilt pathogen to increase to levels that are damaging. Like V. dahliae, F. oxysporum causes a wilt disease by invading the plant’s water conducting tissue, and as with Verticillium wilt, genetic resistance offers a promising approach to disease management. For this reason, we have developed procedures to be used for screening breeding lines and current cultivars for resistance to Fusarium wilt. We also conducted experiments to test for an effect of pH on development of disease, because grower observations suggested that soil acidification might render plants more prone to this problem.

Materials and Methods Assessment of Susceptibility to Verticillium wilt Eleven cultivars and 61 breeding lines were tested for susceptibility to Verticillium wilt. This was accomplished by immersing roots of runner plants in an aqueous suspension of pathogen spores (Gordon et al., 2006). The spore suspension was prepared from plates of potato dextrose agar (PDA) that were fully colonized by a known virulent isolate of V. dahliae. Spores were washed from the plate using sterile distilled water, and the density of spores was adjusted to 29.4 million spores/fluid ounce (= one million spores/ml). Inoculated plants were immediately transplanted into plots at the Wolfskill experimental orchard (Winters, CA) that had been fumigated with 2 methyl bromide:1 chloropicrin at 350 pounds per acre. Two replicate plots of five seedlings per plot were established for each entry. Plants were given a resistance score based on symptoms of disease using a 1 to 5 scale, with 5 corresponding to a healthy plant and 1 corresponding to a severely diseased or dead plant.

Evaluation of Disease Resistance in Naturally Infested Soil In April of 2010, potato tubers were planted at the UC facility in Watsonville, in ground that had been fumigated with 2 methyl bromide:1 chloropicrin at 350 pounds per acre. Stems were inoculated with a spore suspension of V. dahliae in May, and the disease was allowed to develop for approximately three months. In August, stems were cut and moved into a small area (approximately 100 square feet) within the plot and incorporated into the soil, which was tilled periodically to facilitate decomposition. In October, soil was removed from the incorporation area and placed in a mixer to obtain a more uniform distribution of inoculum. After mixing, soil samples were taken and the inoculum density was estimated to be approximately 451.4 ± 11.4 colony forming units per gram of soil (= 15.8 ± 3.5 per gram gram). This Verticillium- infested soil was used to replace approximately one gallon of fumigated soil at each site where a transplant was placed. In this way, nine cultivars and seven breeding lines were established in two replicate beds, with 20 plants per bed. For comparison, 40 plants of each genotype were also subjected to a root-dip inoculation and 40 plants were not inoculated. Disease severity ratings from all three treatments (infested soil, root-dip and control) were taken throughout the 2011 growing season.

11 2011 - 2012 RESEARCH PROJECTS Evaluation of Soil pH on Severity of Fusarium wilt Dormant strawberry plants of the cultivar Camarosa were grown in one gallon pots filled with Sunshine Mix #1 (Sun Gro Horticulture, Canada) that was adjusted to one of four pH levels (pH 5, 6, 7 or 8), and one of four inoculum densities (0; 14,175; 141,750 or 1,417,500 spores per ounce of potting mix or = 500, 5000, and 50 000 spores per gram), for a total of 16 treatments in a factorial design, with four plants (= replications) per treatment. Inoculum for this experiment was produced by growing cultures of F. oxysporum (isolate GL 1080) on PDA for 14 days under ambient lighting at room temperature (20.2 to 22.8 C). Thirty-eight plates were macerated in a blender with 700 ml sterilized de-ionized water, mixed with 6.5 L of twice autoclaved sand, and dried at room temperature. The inoculum density of the sand was estimated using dilution plating on malachite green agar. Inoculated sand was amended with sterile sand as needed to obtain target inoculum densities. Sand mixed with a slurry of non-inoculated PDA and sterilized de-ionized water was used for the control treatment (0 spores /ounce). To obtain the desired pH, phosphoric acid (H3PO4) or sodium hydroxide (NaOH) was added to 2.3 pounds of moistened potting mix as follows: pH 5: 0.5 ounces of 10% H3PO4; pH 6: 0.2 ounces of 1 M NaOH; pH 7: 1.3 ounces of 1 M NaOH; pH 8: 0.2 ounces of 10 M NaOH. Each batch of pH adjusted potting soil was mixed with 1,200 g of sand of the appropriate inoculum density. The inoculated potting mixture was divided among four one gallon pots and placed in a single plastic tray. This process was repeated as needed to obtain the potting mixtures required for all treatments. Soil pH was measured and re-adjusted over the next three days until it remained at the desired level.

Plants were maintained in a growth chamber at day/night temperatures of 77/ 64 F (25/18 C), with a 12 hour photoperiod and supplied with dilute fertilizer (2:1:2 N-P-K in de-ionized water) as needed. Prior to each watering event, a small sample of potting mix from one pot at each inoculum density of each pH level was collected from around the base of the plant. Each sample was suspended in water and the pH was measured. If the pH was more than 0.5 units away from the desired level, either 10% H3PO4 or 1 M NaOH diluted in de-ionized water was added to the pots needing pH adjustment. Soil pH was monitored and adjusted as needed throughout the experiment in this manner. Approximately nine weeks after planting, all plants were rated on a scale of 1 to 5 (with 1 corresponding to a healthy plant and 5 for a plant that died from the disease). The experiment was conducted twice.

A similar experiment to the one described above was conducted using a combination of potting mix and field soil. This experiment included four soil pH levels (pH 5, 6, 7 and 8), three inoculum densities (0, 283,500 and 708,750 spores/ ounce) and two soil treatments (potting mix alone or potting mix combined with field soil at the rate of approximately 30 grams per pot), for a total of 24 treatments in a factorial design, with four plants per treatment. Field soil was collected from a recently fumigated plot at the Wolfskill experimental orchard that was previously cropped to strawberries. Soil was assayed on Komada’s selective medium to confirm the absence ofF. oxysporum f. sp. fragariae. Inoculum was prepared and pH adjusted as described above. Eight weeks after planting, all plants were rated on a scale of 1 to 5, based on symptom development. The experiment was conducted twice.

Efficacy of Fumigation in Eradication of F. oxysporum f. sp. fragariae Inoculum of F. oxysporum f. sp. fragariae was produced by growing the fungus on PDA, blending fully colonized agar in water and adding the resulting slurry to sand. The sand agar mix was stirred periodically over a period over several days until it was dry. Thereafter, the infested sand was packaged in nylon pouches, which were buried at a depth of approximately 6 inches below the surface both at the center and on the shoulder in four replicate beds for each treatment. The following fumigation treatments were applied: 1) Midas EC Gold (33:62) at 169 pounds per acre through drip lines, 2) Midas EC Gold (33:62) at 225 pounds per acre through drip lines, 3) shank applied Pic60 (62% chloropicrin and 38% 1,3 dichloropropene) at 200 pounds per acre, 4) same as treatment #3 but followed by application of 30 gallons per acre of metam sodium through drip lines, 5) shank applied Pic60 at 300 pounds per acre and 6) same as treatment #5 but

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followed by application of 30 gallons per acre of metam sodium through drip lines. Pouches were recovered and the infested sand was assayed by soil dilution plating. In this procedure, sand was suspended in water, and the suspension was spread over the surface of a selective medium. Colonies of F. oxysporum f. sp. fragariae were enumerated in order to quantify the viability of inoculum recovered from each pouch.

Screening for Resistance To Fusarium wilt Ten cultivars and 26 breeding lines (= 36 genotypes) were tested for susceptibility to Fusarium wilt. This was accomplished by immersing roots of runner plants in an aqueous suspension of pathogen spores. A spore suspension was prepared from plates of PDA that were fully colonized by one of five isolates of F. oxysporum f. sp. fragariae. Spores were washed from each plate using sterile distilled water and adjusted by the addition of sterile water to obtain a density of approximately 29.4 million spores/fluid ounce (= one million spores/ml). A spore suspension of each isolate was used to inoculate 400 mL of potato dextrose broth (PDB) in a 1 L flask, which was maintained on a rotary shaker for approximately seven days at room temperature. Colonized PDB from each flask was filtered through three layers of sterilized cheesecloth. The spore density was adjusted to two different levels: 44.1 and 29.4 million spores/fluid ounce by the addition of 0.1% water agar as needed. At both densities, spore suspensions of five isolates were combined so each of the five was represented by approximately 982,00 and 588,000 spores per ounce at the high and low densities, respectively. The high dose five-isolate mix was used to inoculate all 36 genotypes. Four cultivars (‘Albion,’ ‘Ventana,’ ‘San Andreas’ and ‘Camarosa’) were also inoculated with the low dose of the isolate mix and both the high and low doses of a single isolate (GL 1080). Inoculated plants were immediately transplanted into plots that had been fumigated with 2 methyl bromide:1 chloropicrin at 350 pounds per acre at the Wolfskill experimental orchard. Two replicate plots of five seedlings per plot were established for each entry. Plants were given a resistance score based on symptoms of disease using a 1 to 5 scale, with 5 corresponding to a healthy plant and 1 corresponding to a severely diseased or dead plant.

Results Assessment of Susceptibility To Verticillium wilt The eleven cultivars tested in 2010-11 were included mainly to provide a point of reference with previous years. ‘Albion’ and ‘Camino Real’ both had scores of 5.0, whereas ‘Camarosa’ was rated at 4.0. These scores are higher than seen for the same cultivars in most prior years, which is probably due to a weather pattern during 2010-11 that was less favorable to development of Verticillium wilt. Unlike most recently released cultivars, ‘Benicia’ appears to be relatively susceptible to Verticillium wilt, with a resistance score of 3.5. Of the 61 breeding lines tested, resistance scores ranged from 3.0 to 5.0, with an average of 4.6. Sixty-three percent had scores equal to or greater than 4.5 and 44% had the maximum resistance score of 5.0, meaning they were entirely free of symptoms at the end of the season.

Evaluation of Disease Resistance In Naturally Infested Soil The lowest incidence of disease was in the breeding line 94-256-607. This was true for both root dip inoculated plants (1.3%) and plants grown in infested soil (2.5%). The highest incidence was seen in breeding line 4-39-1, with 70% of root-dip inoculated plants and 46% of plants grown in infested soil showing symptoms of Verticillium wilt. Overall, disease incidence was higher in root-dip inoculated plants, for which the average across all genotypes was 23.4%, than in plants grown in infested soil (16.4%). However, the results of the two inoculations methods were strongly and significantly correlated (R2 = 0.893; P < 0.001) (Figure 1).

13 2011 - 2012 RESEARCH PROJECTS 50

45 R2 = 0.893 40 P < 0.001

35

30

25

20

15 10 5

Percentage of symptomatic plants in infested soil 0 0 10 20 30 40 50 60 70 80

Percentage of root-dip inoculated plants symptomatic

Figure 1. Each point corresponds to a single cultivar or breeding line. The location of the point is defined by the percentage of plants showing symptoms of Verticillium wilt when subjected to a root-dip inoculation (X-axis) or when grown in infested soil (Y-axis).

The Effect of pH on Severity of Fusarium wilt in Infested Potting Mix Plants grown in the absence of inoculum (controls) appeared healthy at all pH levels throughout both experiments, whereas disease severity was clearly influenced by inoculum density. On the other hand, there was not an obvious trend in disease development that correlated with soil pH. The final ratings of all plants from both experiments, excluding negative controls, were evaluated using analysis of variance (ANOVA). Experiment (P < 0.001), replication (P < 0.001) and inoculum density (P < 0.001) were significant factors but pH (P = 0.527) was not a significant factor. The effects of several interaction terms were significant (P < 0.050), and consequently data were analyzed separately by experiment.

For the first experiment, the effect of inoculum density on disease severity was significant (P < 0.001), whereas the effects of soil pH (P = 0.408) and replication (P = 0.130) were not. The pH by inoculum density interaction was significant (P < 0.001), so data were analyzed separately by inoculum density. At the low inoculum level (14,175 spores /ounce), the effect of pH was significant (P < 0.001), whereas replication was not (P = 1.000). Mean disease severity ratings (± standard error) were: 2.0 ± 0.0 at pH 5, 1.3 ± 0.1 at pH 6, 1.5 ± 0.0 at pH 7, and 2.3 ± 0.3 at pH 8. At the intermediate inoculum level (141,750 spores /ounce), replication was significant (P < 0.001) and pH was not (P = 0.127). Mean disease severity ratings were: 2.3 ± 0.3 at pH 5, 2.3 ± 0.2 at pH 6, 3.6 ± 0.4 at pH 7, and 2.3 ± 0.8 at pH 8. At the high inoculum density (1,417,500 spores /ounce), the effects of pH (P = 0.270) and replication (P = 0.987) were both not significant. Mean disease severity ratings for plants grown at this inoculum density were: 4.9 ± 0.1 at pH 5, 5.0 ± 0.0 at pH 6, 4.8 ± 0.3 at pH 7, and 4.5 ± 0.3 at pH 8 (Figure 2).

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6 pH 5

5 pH 6

pH 7 4 pH 8

3

2

Disease severity rating 1

0 Low Intermediate High Inoculum density

Figure 2. Severity of disease on a 1 to 5 scale (with 1 corresponding to a healthy plant and 5 corresponding to a plant killed by Fusarium wilt) for the cultivar ‘Camarosa’ grown in potting mix with three different levels of inoculum (low, intermediate and high) and at four different levels of soil pH (5, 6, 7 and 8). This figure presents the results of the first experiment.

Based on data from the second experiment, the effect of inoculum density was significant (P < 0.001), whereas the effects of soil pH (P = 0.305) and replication (P = 0.136) were not significant. The pH by inoculum density interaction was significant (P = 0.018), and consequently data were analyzed separately by inoculum density. At 14,175 spores / ounce, pH (P = 0.007) and replication (P = 0.028) were significant. Mean disease severity ratings for plants grown at this inoculum density were: 1.1 ± 0.1 at pH 5, 2.8 ± 0.8 at pH 6, 2.8 ± 0.3 at pH 7, and 3.6 ± 0.4 at pH 8. At 141,750 spores /ounce, the effects of pH (P = 0.045) and replication (P < 0.001) were significant. Mean disease severity ratings for plants grown at 141,750 spores /ounce were: 3.9 ± 0.7 at pH 5, 4.4 ± 0.6 at pH 6, 4.5 ± 0.5 at pH 7, and 3.1 ± 0.7 at pH 8. At 1,417,500 spores /ounce, the effect of pH was significant (P < 0.001) but replication was not (P = 0.972). Mean disease severity ratings were: 5.0 ± 0.0 at pH 5, 5.0 ± 0.0 at pH 6, 4.1 ± 0.1 at pH 7, and 4.4 ± 0.2 at pH 8 (Figure 3).

Figure 3. Severity of disease on a 1 to 5 scale (with 1 corresponding to a healthy plant and 5 corresponding to a plant killed by Fusarium wilt) for the cultivar ‘Camarosa’ grown in potting mix with three different levels of inoculum (low, intermediate and high) and at four different levels of soil pH (5, 6, 7 and 8). This figure presents the results of the second experiment.

15 2011 - 2012 RESEARCH PROJECTS Effect of pH on Disease Severity in Field Soil All plants grown in non-inoculated soil remained free of Fusarium wilt symptoms throughout the course of both experiments. At the high inoculum level (708,750 spores/ounce), all plants developed severe symptoms of Fusarium wilt and there was extensive mortality, regardless of pH or soil composition. At the low inoculum level (283,500 spores/ounce), plants grown in potting mix combined with field soil often had slightly higher disease severity ratings than those grown in potting mix alone (Figure 4). However, based on data from both experiments at the lower inoculum level, ANOVA showed pH to be the only significant factor (P = 0.039). Experiment (P = 0.211), inoculum density (P = 0.215), treatment (field soil present or absent, P = 0.554), and replication (P = 0.054) were all not significant. Likewise, all interactions between these factors were not significant (P ≥ 0.198). Mean disease severity ratings averaged across treatment were 2.7, 3.5, 4.0 and 4.0 for plants grown at pH 5, 6, 7 and 8.

Figure 4. Severity of disease on a 1 to 5 scale (with 1 corresponding to a healthy plant and 5 corresponding to a plant killed by Fusarium wilt) for the cultivar ‘Camarosa’ grown in potting mix with and without field soil at four different pH levels (5, 6, 7 and 8).

Effect of Fumigation in Eradication of F. oxysporum f. sp. fragariae Fusarium oxysporum f. sp. fragariae was not detectable in infested sand buried at the center of beds treated with Midas EC Gold at either 169 or 225 pounds per acre, but 44,192 spores per ounce were recovered from pouches buried at the side of the bed, where the low rate was applied. Both rates of shank-applied Pic60 eliminated the pathogen at the sides of the bed but the pathogen remained viable at the center of the bed, with 10,894 and 1,210 spores per ounce surviving at the low (200 pounds per acre) and high rates (300 pounds per acre), respectively. Where the low rate of Pic60 was followed by 30 gallons per acre of metam sodium, surviving inoculum densities were 303 and 9,104 spores per ounce at the center and side of the bed, respectively. The high rate of Pic60 followed by metam sodium eliminated the pathogen at the center of the bed but 605 spores per ounce survived at the side of the bed.

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Screening for Resistance to Fusarium wilt Disease developed as expected, with susceptible cultivars such as ‘Camarosa’ and ‘Albion’ displaying severe symptoms by the final rating, whereas the resistant cultivar, ‘Ventana’, showed at most mild stunting, regardless of treatment. The higher inoculation dose (982,000 spores per ounce) tended to result in more severe disease than the lower dose (588,000 spores per ounce). For example, resistance scores for ‘Albion’ inoculated with GL1080 were 3.0 and 1.5 at the low and high doses, respectively. However, analysis of the full data set showed the effect of dose on disease resistance scores not to be significant (P = 0.077). Likewise the effect of isolate (P = 0.141) and all interactions (P ≥ 0.184) were not significant. When non-significant terms were removed from the model, differences among genotypes were significant (P < 0.001). Resistance scores for 26 breeding lines ranged from 1.0 to 5.0, with a mean of 3.3.

Discussion Scores for resistance to Verticillium wilt among UC breeding lines are slightly above what was recorded in 2010. Sixty-three percent had scores at or above 4.5 as compared to 56% last year. However, year over year comparisons of the same genotypes indicate that disease severity was lower overall in 2011. Consequently, the actual differences in susceptibility between breeding lines in the two years are probably less than what is implied by data from 2011. Although most recently released cultivars are significantly more resistant than those developed prior to adoption of the current screening protocol, ‘Benicia’ appears to be an exception, based on a relatively low resistance score in the 2011 test.

Tests conducted in soil naturally infested with V. dahliae were intended to assess the performance of cultivars and breeding lines that have been selected based on their reaction to artificial inoculations. The 2011 experiment was a repeat of a similar experiment conducted in 2010. In 2010, plants were exposed to approximately 300 microsclerotia per ounce of soil, whereas in the 2011 tests inoculum density was estimated to be about 450 microsclerotia per ounce. For comparison, something in the range of 100 microsclerotia per ounce of soil would be expected to cause a significant economic loss in most years. In both 2010 and 2011 there was a strong and highly significant correlation between resistance scores observed for plants subjected to root-dip inoculations and those becoming infected by exposure to soilborne inoculum. This indicates that the screening procedure being used by the UC breeding program has been effective in identifying differences in susceptibility that correspond with how strawberry genotypes respond to the disease under natural conditions.

Previous work has shown that liming to bring soil pH to 6.5 and above can reduce severity of Fusarium wilt affecting watermelon (Everett and Blazquez, 1967) and tomato (Jones and Woltz, 1967). This effect has been attributed to greater availability of micro-nutrients for the pathogen under acidic conditions and more intense competition with bacteria when soil pH is closer to neutral. Grower observations suggested that Fusarium wilt of strawberry in California was more commonly seen in fields where fertigation (the application of nutrient solutions through drip irrigation lines) acidified soil. Whereas growers have found this practice beneficial because lowering soil pH makes micro-nutrients more available to plants, it may also enhance disease severity by releasing the Fusarium wilt pathogen from nutrient limitations on growth and infection.

17 2011 - 2012 RESEARCH PROJECTS The results of our experiments showed that the effect of pH on severity of Fusarium wilt was significant at some inoculum levels when plants were grown in potting mix. At the highest inoculum level disease was severe in all treatments so it was not possible to assess an effect of soil pH. At the lowest inoculum level, disease was relatively mild and only small differences in disease were observed. The most conspicuous effect of pH was apparent at the intermediate inoculum level, with disease being less severe at pH 5.0 than at 7.0 or 8.0. This is the opposite of what would be expected based on published results of pH effects on Fusarium wilt diseases affecting other crops. Of course, potting mix lacks the usual complement of bacteria found in soil and their absence may obscure an effect that would be evident under field conditions. However, even where field soil was included with potting mix, disease severity on average was lowest at pH 5.0 and greatest at pH 7.0 and pH 8.0.

It is not clear why our findings differ from what has been reported for Fusarium wilt of watermelon and tomato. It may reflect differences in the causal pathogens and/or conditions under which tests were conducted. However, it is also important to note that there are conflicting results in the literature regarding the effect of pH on development of Fusarium wilt in watermelon. In a study conducted in a naturally infested field, increased soil pH did not reduce disease severity (Hopkins and Elmstrom, 1976). Notwithstanding these negative results and our own findings under controlled conditions, we cannot exclude the possibility that soil pH affects severity of Fusarium wilt of strawberry under some circumstances. However, even if this is so, it seems unlikely that the effect of pH is strong enough to be a determinative factor in disease development in most fields, which precludes making any general recommendations on adjustment of soil pH for disease management.

Our fumigation experiment documented a differential effect of bed location on efficacy where fumigant is applied through drip lines. The lesser reduction at the side as opposed to the center of the bed is consistent with results of previous experiments and underscores the need to use fumigation practices that will maximize distribution within the bed. This may include using additional drip lines and/or increasing the volume of water used to deliver the fumigant. Where fumigants were applied by shank injection, efficacy tended to be lower at the center of the bed than on the sides. This result indicates that shank injection may also fail to achieve adequate distribution of fumigant within a bed.

As in 2010, the results of screening for resistance to Fusarium wilt showed some cultivars and breeding lines to be highly resistant to Fusarium wilt. ‘Ventana’ and ‘San Andreas’ both fall into the resistant category and this classification is supported by previous field trials where both cultivars were exposed to high inoculum levels. Conditions during our 2011 test were conducive to disease development as indicated by severe disease affecting both ‘Albion’ and ‘Camarosa.’ A significant number of breeding lines appeared to be highly resistant to Fusarium wilt, with 6 of 26 tested lines (23%) receiving the maximum score of 5.0 However, five breeding lines (19%) had resistance scores of 1.5 or less, indicating that considerable variation in susceptibility remains within the UC breeding population.

We have continued to monitor the occurrence of Fusarium wilt and charcoal rot, caused by M. phaseolina, within California. Charcoal rot was originally found in only two counties (Orange and Ventura), but has now been confirmed to occur in all major strawberry production regions in the state, including fields in Alameda, Contra Costa, Los Angeles, Monterey, Orange, Sacramento, San Diego, San Luis Obispo, Santa Barbara, Santa Clara, Santa Cruz, Ventura and Yolo Counties. Fusarium wilt of strawberry was originally limited to Ventura County. However, in early 2012, we isolated what appears to be the Fusarium wilt pathogen from declining strawberry plants in San Luis Obispo and Santa Cruz counties. These isolates cannot be considered pathogenic until further testing (inoculation tests) is completed. The Gordon and Koike labs are currently working to confirm pathogenicity of these isolates. In each succeeding year since their initial discoveries, the number of ranches infested with these two pathogens has continued to increase. In most cases, the disease is being found in fields where standard pre-plant, flat fumigation application of methyl bromide + chloropicrin has not been used.

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Selected References

• Everett P.H. and C.H. Blazquez. 1967. Influence of lime on the development of Fusarium wilt of watermelons. Florida Agricultural Experiment Stations Journal Series 2845:143-148.

• Gordon T.R., Kirkpatrick S.C., Hansen J. and D.V. Shaw. 2006. The response of strawberry genotypes to inoculation with isolates of Verticillium dahliae differing in host origin. Plant Pathology 55:766-769.

• Hopkins D.L. and G.W. Elmstrom. 1976. Effect of soil pH and nitrogen source on Fusarium wilt of watermelon on land previously cropped in watermelons. Proceedings of the Florida State Horticultural Society 89:141-143.

• Jones J.P. and S.S. Woltz. 1967. Fusarium wilt (race 2) of tomato: Effect of lime and micronutrient soil amendments on disease development. Plant Disease Report 51:645-648.

• Koike S.T., Kirkpatrick S.C. and T.R. Gordon. 2009. Fusarium wilt of strawberry caused by Fusarium oxysporum in California. Plant Disease 93:1077-1077.

• Shaw D.V., Gordon T.R., Larson K.D., Gubler W.D., Hansen, J. and S.C. Kirkpatrick. 2010a. Genetic progress in breeding for resistance of strawberry (Fragaria x ananassa Duch.) to Verticillium wilt (Verticillium dahliae Kleb.) California Agriculture 64:37-41.

• Shaw D.V., Gordon T.R., Hansen J. and S.C. Kirkpatrick. 2010b. Relationship between the extent of colonization by Verticillium dahliae and symptom expression in strawberry (Fragaria x ananassa) genotypes resistant to Verticillium wilt. Plant Pathology, 59:376-381.

19 2011 - 2012 RESEARCH PROJECTS