Botrytis fruit rot or gray mold, caused by Botrytis cinerea, is common fruit disease in California strawberries (Koike et al. 2018).  Botrytis cinerea has a wide host range infecting several commercially important crops including blueberry (Saito et al. 2016), grapes (Saito et al., 2019), and tomato (Breeze, 2019).  Fungal infection can cause flower or fruit rot.  Fruit can be infected directly or through a latent infection in the flowers.  Moist and cool conditions favor fungal infections and increased sugar content in the ripening fruit can also contribute to the disease development.  Initial symptoms of infection appear as brown lesions and a thick mat of gray conidia is characteristic symptom in the later stages of infection.  As chemical fungicides are primarily used for gray mold control, fungicide resistance is a common problem around the world (Panebianco et al., 2015; Liu et al., 2016; Stockwell et al., 2018; Weber and Hahn, 2019).  In strawberry, cultural control options such as removing diseased plant material or using cultivars with traits that can reduce gray mold infections may not be practical when the disease is widespread in the field or cultivar choice is made based on other factors.  Non-chemical control options are necessary to help reduce the risk of chemical fungicide resistance, prolong the life of available chemical fungicides, achieve desired disease control, and to maintain environmental health.  Although there are several botanical and microbial fungicides available for gray mold control, limited information is available on their efficacy in California strawberries.  A study was conducted in the spring of 2019 to evaluate the efficacy of several chemical, botanical, and microbial fungicides in certain combinations and rotations to help identify effective options for an integrated disease management strategy.

Methodology

Strawberry cultivar San Andreas was planted late November, 2018 and the study was conducted in April and May, 2019.  Each treatment had a 20' long strawberry plot with two rows of plants replicated in a randomized complete block design.  Plots were maintained without any fungicidal applications until the study was initiated.  Table 1 contains the list of treatments, application rates and dates of application, and Table 2 contains the type of fungicide used and their mode of action.  Beauveria bassiana and Metarhizium anisopliae s.l. are California isolates of entomopathogenic fungi, isolated from an insect and a soil sample, respectively.  These fungi are pathogenic to a variety of arthropods and some strains are formulated as biopesticides for arthropod control.  However, earlier studies in California demonstrated that these fungi are also known to antagonize plant pathogens such as Fusarium oxysporum f.sp. vasinfectum Race 4 (Dara et al., 2016) and Macrophomina phaseolina (Dara et al., 2018) and reduce the disease severity.  To further evaluate their efficacy against B. cinerea, these two fungi were also included in this study alternating with two chemical fungicides.

Treatments were applied with a CO₂-pressurized backpack sprayer using 66.5 gpa spray volume.  Five days before the first spray application and 3 days after each application, all ripe fruit were harvested from each plot and incubated at the room temperature in vented plastic containers.  The level of gray mold on fruit from each plot was rated using a 0 to 4 scale (where 0=no disease, 1=1-25% fruit with fungal infection, 2=26-50% infection, 3=51-75%, and 4=76-100%) 3 and 5 days after each harvest (DAH).  Due to the rains, fruit could not be harvested after the 3^rd spray application for disease rating, but was harvested and discarded after the rains to avoid cross infection for the following week's harvest.  Data were analyzed using analysis of variance using Statistix software and significant means were separated using Least Significant Difference separation test.

Results

Gray mold occurred at low to moderate levels during the study period.  Along with B. cinerea, there were a few instances of minor fungal infections from Rhizopus spp. (Rhizopus fruit rot) and Mucor spp. (Mucor fruit rot).  Pre-treatment disease ratings were statistically not significant (P = 0.6197 and 0.5741) 3 and 5 DAH.  While the chemical standard treatment with the rotation of Captan, Merivon, Switch, and Pristine (treatment 2) appeared to result in the lowest disease rating throughout the observation period, treatments 3 and 5 after the 1^st spray application, treatments 5 and 11 along with 3, 4 and 6 after the 2^nd spray application, and treatments 3 and 5 along with 11 after the 4^th spray application also had similar disease control at 3 DAH.  When disease at 5 DAH was compared, the lowest rating was seen in treatment 2 after the 1^st and 2^nd spray applications, and treatments 2, 3, and 11 after the 4^th application.  Several other treatments also provided statistically similar control during these days.

When the average disease rating for the three post-treatment observation events was considered, treatment 2, 3, 5, and 11 had the lowest disease at both 3 and 5 DAH.  Treatments 4 and 12 at 3 DAH also had a statistically similar level of disease control to treatment 2. 

In general, most of the treatments provided moderate to high control compared to the disease in untreated control when the post-treatment averages were considered.  Only treatment 7 and 13 had lower control at 3 DAH.

Discussion

This study compared a variety of registered and developmental products along with two entomopathogenic fungi in managing B. cinerea.  Considering the fungicide resistance problem in B. cinerea in multiple crops, having multiple non-chemical control options is very important to achieve desirable control with integrated disease management strategies.  Since the active ingredients in the botanical and bacterial fungicides used in this study are not public, discuss will be limited on their modes of action and efficacy at this point.  Similarly, the active ingredient of WXF-17001 is also not known, however, an earlier study by Calvo-Garrido et al. (2014) demonstrated that a fatty acid-based natural product reduced B. cinerea conidial germination by 54% and disease severity in grapes by 96% compared to untreated control.  The product used by Calvo-Garrido et al. (2014) is thought to be fungistatic and reduce the postharvest respiratory activity and ethylene production in fruits.

While chemical fungicides have a specific mode of action, biological and other products act in multiple manners either directly antagonizing the plant pathogen or by triggering the plant defenses.  For example, amending the potting medium with biochar resulted in induced systemic resistance in tomato and reduced B. cinerea severity by 50% (Mehari et al., 2015).  Luna et al. (2016) also showed that application of β-aminobutyric acid and jasmonic acid promoted seed germination and long-term resistance to B. cinerea in tomato.  Burkholderia phytofirmans, beneficial endophytic bacterium, offered protection against B. cinerea in grapes by mobilizing carbon resources (callose deposition), triggering plant immune system (hydrogen peroxide production and priming of defense genese), and through antifungal activity (Miotto-Vilanova et al. 2016).  Similarly, entomopathogenic fungi such as B. bassiana are also known to induce systemic resistance against plant pathogens (Griffin et al. 2006).  Compared to other options evaluated in the study, entomopathogenic fungi have an advantage of controlling both arthropod pests and diseases, while also having plant growth promoting effect (Dara et al. 2017).

Rotating fungicides with different mode of actions reduces the risk of resistance development and using some combinations will also maintain control efficacy.  This study provided the efficacy of multiple control options and their combinations and rotations for B. cinerea.  This is also the first study demonstrating the efficacy of entomopathogenic fungi against B. cinerea in strawberry.

Acknowledgements: Thanks to Sipcam Agro and Westbridge for funding the study, technical assistance of Hamza Khairi for data collection, and the field staff at the Shafter Research Station for the crop maintenance.

References

Breeze, E.  2019.  97 Shades of gray: genetic interactions of the gray mold, Botrytis cinerea, with wild and domesticated tomato.  The Plant Cell 31: 280-281.  https://doi.org/10.1105/tpc.19.00030

Calvo-Garrido, C., A.A.G. Elmer, F. J. Parry, I. Viñas, J. Usall,  R. Torres,  R.H. Agnew, and  N. Teixidó.  2014.  Mode of action of a fatty acid-based natural product to control Botrytis cinerea in grapes.  J. Appl. Microbiol. 116: 967-979.  https://doi.org/10.1111/jam.12430

Dara, S. K., S. S. Dara, S.S.R. Dara, and T. Anderson.  2016.  First report of three entomopathogenic fungi offering protection against the plant pathogen, Fusarium oxysporum f.sp. vasinfectum.  UC ANR eJournal of Entomology and Biologicals https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=22199  

Dara, S. K., S.S.R. Dara, and S. S. Dara.  2017.  Impact of entomopathogenic fungi on the growth, development, and health of cabbage growing under water stress.  Amer. J. Plant Sci. 8: 1224-1233.  https://doi.org/10.4236/ajps.2017.86081

Dara, S.S.R., S. S. Dara, and S. K. Dara.  2018.  Preliminary report on the potential of Beauveria bassiana and Metarhizium anisopliae s.l. in antagonizing the charcoal rot causing fungus Macrophomina phaseolina in strawberry.  UC ANR eJournal of Entomology and Biologicals https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=28274

Griffin, M. R., B. H. Ownley, W. E. Klingeman, and R. M. Pereira.  2006.  Evidence of induced systemic resistance with Beauveria bassiana against Xanthomonas in cotton.  Phytopathol. 96.

Koike, S. T., G. T. Browne, T. R. Gordon, and M. P. Bolda.  2018.  UC IPM pest management guidelines: strawberry (diseases).  UC ANR Publication 3468. https://www2.ipm.ucanr.edu/agriculture/strawberry/Botrytis-Fruit-Rot/

Liu, S., Z. Che, and G. Chen.  2016.  Multiple-fungicide resistance to carbendazim, diethofencardb, procymidone, and pyrimethanil in field isolates of Botrytis cinerea from tomato in Henan Province, China.  Crop Protection 84: 56-61.

Luna, E., E. Beardon, S. Ravnskov, J. Scholes, and J. Ton.  2016.  Optimizing chemically induced resistance in tomato against Botrytis cinerea.  Plant Dis. 100: 704-710. https://doi.org/10.1094/PDIS-03-15-0347-RE

Mehari, Z. H., Y. Elad, D. Rav-David, E. R. Graber, and Y. M. Harel.  2015.  Induced systemic resistance in tomato (Solanum lycopersicum) against Botrytis cinerea by biochar amendment involves jasmonic acid signaling.  Plant and Soil 395: 31-44.

Miotto-Vilanova, L., C. Jacquard, B. Courteaux, L. Wortham, J. Michel, C. Clément, E. A. Barka, and L. Sanchez.  2016.  Burkholderia phytofirmans PsJN confers grapevine resistance against Botrytis cinerea via a direct antimicrobial effect combined with a better resource mobilization.  Front. Plant Sci. 7: 1236. https://doi.org/10.3389/fpls.2016.01236

Panebianco, A., I. Castello, G. Cirvilleri, G. Perrone, F. Epifani, M. Ferrarra, G. Polizzi, D. R. Walters, and A. Vitale.  2015.  Detection of Botrytis cinerea field isolates with multiple fungicide resistance from table grape in Sicily.  Crop Protection 77: 65-73.

Saito, S., T. J. Michailides, and C. L. Xiao.  2016.  Fungicide resistance profiling in Botrytis cinerea populations from blueberry in California and Washington and their impact on control of gray mold.  Plant Dis. 100: 2087-2093. https://doi.org/10.1094/PDIS-02-16-0229-RE

Saito, S., T. J. Michailides, and C. L. Xiao.  2019.  Fungicide-resistant phenotypes in Botrytis cinerea populations and their impact on control of gray mold on stored table grapes in California.  European J. Plant Pathol. 154: 203-213.

Stockwell, V. O., B. T> Shaffer, L. A. Jones, and J. W. Pscheidt.  2018.  Fungicide resistance profiles of Botrytis cinerea isolated from berry crops in Oregon.  Abstract for International Congress of Plant Pathology: Plant Health in A Global Economy; 2018 July 29-Aug 3; Boston, MA.

Weber, R.W.S. and M. Hahn.  2019.  Grey mould disease of strawberry in northern Germany: causal agents, fungicide resistance and management strategies.  Appl. Microbiol. Biotechnol. 103: 1589-1597.

Transplanting tomatoes

There has been a growing interest in the recent years in exploring the potential of biostimulants in crop production.  Biostimulants are mineral, botanical, or microbial materials that stimulate natural processes in plants, help them tolerate biotic and abiotic stressors, and improve crop growth and health.  Several recent studies demonstrated the potential of the biostimulant or soil amendments in improving crop yields and health.  For example, in a 2017 field study, silicon, microbial, botanical and nutrient materials improved processing tomato yields by 27 to 32% compared to the standard fertility program (Dara and Lewis, 2018).  In a 2017-2018 strawberry field study, some biostimulant and soil amendment products resulted in a 13-16% increase in marketable fruit yield compared to the grower standard (Dara and Peck, 2018).  He et al. (2019) evaluated three species of Bacillus and Pseudomonas putida alone and in different combinations in tomatoes grown in laboratory and greenhouse.  The combination of Bacillus amyloliquefaciens, B. pumilus, and P. putida increased the plant biomass and the root/shoot ratio.  Significant increase in fruit yield, between 18 and 39%, was also achieved from individual or co-inoculations of these bacteria.  A field study was conducted in processing tomato to evaluate the impact of nutrient products containing beneficial microbes and botanical extracts on tomato yields and fruit quality.

Tomato plots - each treatment is marked by a colored flag

Methodology

The study was conducted from late spring to fall of 2018 to evaluate three treatment programs compared to the grower standard.  Tomato cultivar Quali T27 was seeded on 25 April and transplanted on 19 June using a mechanical transplanter.  Due to high temperatures at the time of planting, some transplants died and they were re-planted on 28 June.  Herbicide Matrix was applied on 5 July and Poast was applied on 13 July followed by hand weeding on 27 July.  Crop was irrigated, fertigated, and treatements were applied through a drip system.  Overhead sprinkler irrigation was additionally used immediately after transplanting.  The following treatments were included in the study:

1. Grower standard: 10-34-0 Ammonium Polyphosphate Solution was applied at 10 gal/ac at the time of transplanting followed by the application of UAN-32 Urea Ammonium Nitrate Solution 32-0-0 at the rate of 15 units of N at 3, 6, and 13 weeks after planting and 25 units of N at 7 weeks after planting.

2. Grower standard + BiOWiSH Crop 16-40-0: BiOWiSH Crop 16-40-0 contains 16% nitrogen and 40% phosphate along with B. amyloliquefaciens, B. licheniformis, B. pumilus, and B. subtilis at 1X10⁸ cfu/gram.  Crop 16-40-0 was applied at 1 lb/ac at the time of planting followed by the application 0.5 lb/ac at 3, 6, and 9 weeks after planting.

3. Grower standard 85% + BiOWiSH Crop 16-40-0: Crop 16-40-0 was applied at the same rate and frequency as in treatment 2, but the grower standard was reduced to 85%.

4. RootRx: RootRx contains 5% soluble potash and proprietary botanical extracts and is supposed to stimulate a broad range of antioxidant compounds in the plant.  It was applied at 0.25 gal/ac at the time of planting followed by the application of 0.5 gal/ac at 3, about 7, and 13 weeks after planting.

Each treatment contained 30' long bed with a single row of tomato plants and replicated five times in a randomized complete block design.  Along with the fruit yield, the sugar content of the fruit and leaves [using a refractometer from three fruits (two measurements from each) and four leaves per plot], chlorophyll content (using a digital chlorophyll meter from four leaves per plot), and frost damage levels (using a visual rating on a 0 to 5 scale where 0 = no frost damage and 5 = extreme frost damage with a complete plant death) were also monitored.  Due to an unknown reason, some plants in the fifth replication were stunted halfway through the study.  Data from the fifth replication were excluded from the analysis.  Data were subjected to the analysis of variance using Statistix software and significant means were separated using the Tukey's HSD test. 

Results

Fruit yield: Marketable and unmarketable fruit yield was monitored from 27 August to 13 November.  Seasonal total for marketable fruit was significantly (P = 0.04) different among the treatments where RootRx resulted in a 26.5% increase over the grower standard while Crop 16-4-0 with the full rate of the grower standard had an 8%, and with 85% of the grower standard had a 13.2% increase.  It appeared that a similar improved yield response was also seen when Crop 16-40-0 was used at a reduced rate of the grower standard in other studies conducted by the manufacturer.

Sugar content: Sugar content of the fruit and leaves was measured once after the last harvest and there were no significant (P > 0.05) difference among the treatments.

Chlorophyll content: Chlorophyll content was measured once after the last harvest and there was no significant (P > 0.05) difference among the treatments.

Frost damage: Study was concluded after frosty conditions in November 2018 damaged the crop.  Although there were no statistically significant (P > 0.05) differences, plants treated with RootRx had the lowest rating of 2.

Means and standard errors of the measured parameters

Acknowledgements: Thanks to Jenita Thinakaran and the field staff at the Shafter Research Station for their technical assistance, Plantel Nurseries for providing transplants, and BiOWiSH Technologies and Redox Chemicals for their financial support.

References

Dara, S. K. and D. Peck.  2018.  Microbial and bioactive soil amendments for improving strawberry crop growth, health, and fruit yields: a 2017-2018 study.  UCANR eJournal of Entomology and Biologicals (https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=27891)

Dara, S. K. and E. Lewis.  2018.  Impact of nutrient and biostimulant materials on tomato crop health and yield.  UCANR eJournal of Entomology and Biologicals (https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=26054)

He. Y., H. A. Pantigoso, Z. Wu, and J. M. Vivanco.  2019.  Co-inoculation of Bacillus sp. and Pseudomonas putida at different development stages acts as a biostimulant to promote growth, yield and nutrient uptake of tomato.  J. Appl. Microbiol.  https://doi.org/10.1111/jam.14273

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The western Grapeleaf skeletonizer (WGLS), Harrisina metallica Stretch (Lepidoptera: Zygaenidae), previously known to cause severe defoliation to vineyards and backyard grapevines appears to be re-emerging in California.  Since its first detection in San Diego in 1941, WGLS spread through commercial vineyards and backyard grapes becoming a serious problem.  Although two biological control agents from Arizona and Mexico were introduced in California for WGLS control, a naturally occurring granulovirus (Harrisina brillians granulovirus) nearly eradicated WGLS populations and kept them under control.  WGLS has not been a problem especially in conventional vineyards.  However, based on some unpublished observations, WGLS populations are emerging in organic vineyards and backyard grapevines.

WGLS lives up to its name by skeletonizing and defoliating grape leaves. Organic vineyards are especially at risk and uncontrolled populations can destroy vineyards resulting in significant losses. Metallic bluish or greenish black moths lay barrel shaped yellowish eggs on the lower side of the leaves.  There are five larval instars.  Early instars are cream colored and develop black and purple bands in later stages.  Pupation occurs in a whitish cocoon.  Upon hatching, larvae start feeding side by side in a row on the lower side of leaf.  Damage by younger larvae appears as whitish leaf area containing veins and the upper cuticle, which eventually turn brown.  Older larvae skeletonize leaves leaving larger veins.  Larvae may also feed on fruit leading to bunch rot.  Severe damage can cause defoliation and sunburn of the exposed fruit. 

Methodology

A study was conducted to evaluate the efficacy of six non-chemical control options that included formulations of spinosad, two subspecies of Bacillus thuringiensis, and a botanical insecticide/growth regulator along with two unformulated entomopathogenic fungal isolates native to California.  Larvae were collected from an infested, untreated backyard grapevine and maintained in one gallon plastic tubs with screened lids on infested leaves.  Fresh, untreated grape leaves from uninfested vines were provided daily for 3 days before starting the assay.  For each treatment, five 4-5 instar larvae were placed on a grape leaf disc (rinsed in water and dried) in a Petri plate (100 mm dia) with a moist filter paper.  Larvae were treated by spraying 1 ml of the treatment solution (containing Dyne-Amic as a surfactant at 0.125% vol/vol).  Application rates for commercial formulations were determined based on label recommendations for 100 gallons of spray volume.  Entomopathogenic fungal concentrations were also determined based on the label rates for similar commercial products. Treatments were replicated four times and the assay was conducted twice.  Larval mortality was observed daily and dead larvae were removed and incubated separately.  Fresh leaf discs were provided as needed to the remaining larvae.  Actual and corrected (for control mortality) total mortality were calculated.Data were arcsine-transformed for statistical analysis and significant means were separated using Tukey's HSD test. 

Results

Both cumulative daily mortality and total mortality significantly (P < 0.0001) differed among treatments. Entrust and M. anisopliae resulted in the highest mortality followed by B. bassiana, Neemix, and Agree. In general, feeding reduced or ceased in all larvae following treatment and could have contributed to a lower mortality in B. thuringiensis treatments.  Entomopathogenic fungi emerged from all the cadavers from respective treatments.  Microbial and botanical options provided good control of WGLS.  These non-chemical alternatives can be effectively used in both organic and conventional vineyards.  California isolates of B. bassiana and M. anisopliae demonstrated good control efficacy and the potential to be developed as microbial pesticides.

Healthy larva in untreated control and those killed by different treatments

Acknowledgements: Thanks to the technical assistance of Alor Sahoo in carrying out these assays, and Certis and Corteva for providing the pesticide formulations.

References

Federici, B. A. and V. M. Stern.  1990.  Replication and occlusion of a granulosis virus in larval and adult midgut epithelium of the western grapeleaf skeletonizer, Harrisina brillians.  J. Invertebr. Pathol. 56: 401-414.

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Potted strawberry plants color-coded for different treatments.  Photo by Surendra Dara

Charcoal rot, caused by Macrophomina phaseolina, is one of the important fungal diseases of strawberry in California.  Macrophomina phaseolina is a soilborne fungus and has a wide host range, including alfalfa, cabbage, corn, pepper, and potato, some of which are cultivated in the strawberry production areas in California.  The fungus infects the vascular system of the plant roots, obstructing the nutrient and water supply and ultimately resulting in stunted growth, wilting, and death of the plant.  The fungus survives in the soil and infected plant debris as microsclerotia (resting structures made of hyphal bodies) and can persist for up to three years.  Microslerotia germinate and penetrate the root system to initiate infection.  Plants are more vulnerable to fungal infection when they are experiencing environmental (extreme weather or drought conditions) and physiological (heavy fruit bearing) stress.

Soil fumigation is the primary management option for addressing charcoal rot in strawberry.  Crop rotation with broccoli can also reduce the risk of charcoal rot due to glucosinolates and isothiocyanates in broccoli crop residue that have fungicidal properties.  Beneficial microorganisms such as Bacillus spp. and Trichoderma spp. are also considered, especially in organic strawberries, to antagonize M. phaseolina and other soilborne pathogens and provide some protection.  The role of beneficial microbes in disease management or improving crop growth and health is gaining popularity in the recent years with the commercial availability of biofungicide, biostimulant, and soil amendment products.  In a couple of recent strawberry field studies in Santa Maria, some of the beneficial microbial products improved fruit yield or crop health.  These treatments can be administered by inoculating the transplants prior to planting, immediately after planting or periodically applying to the plants and or the soil.  Adding beneficial microbes can help improve the soil microbiome especially after chemical or bio-fumigation and anaerobic soil disinfestation. 

Similar to the benefits of traditionally used bacteria (e.g., Bacillus spp. and Pseudomonas spp.) and fungi (e.g., Glomus spp. and Trichoderma spp.), studies with entomopathogenic fungi such as Beauveria bassiana, Isaria fumosorosea, and Metarhizium spp. also demonstrated their role in improving water and nutrient absorption or antagonizing plant pathogens.  The advantage of entomopathogenic fungi is that they are already used for arthropod pest management in multiple crops, including strawberry; now, there are the additional benefits of promoting crop growth and antagonizing plant pathogens.  In light of some promising recent studies exploring these roles, a study was conducted using potted strawberry plants to evaluate the efficacy of two California isolates of Beauveria bassiana and Metarhizium anisopliae s.l. and their application strategies against M. phaseolina.

Methodology

About 6 week old strawberry plants (cultivar Albion) from a strawberry field at the Shafter Research Station were transplanted into 1.6-gallon pots with Miracle-Gro All Purpose Garden Soil (0.09-0.05-0.07 N-P-K) and maintained in an outdoor environment.  They were regularly watered, and their health was monitored for about 5 months prior to the commencement of the study.  Conidial suspensions of the California isolates of B. bassiana and M. anisopliae s.l. were applied one week before, after, or at the time of applying microsclerotia of M. phaseolina to the potting mix.  The following treatments were evaluated in the study:

1. Untreated control
2. Soil inoculated with M. phaseolina
3. Soil inoculated with  B. bassiana 1 week prior to M. phaseolina inoculation
4. Soil inoculated with  M. anisopliae s.l. 1 week prior to M. phaseolina inoculation
5. Soil inoculated with  B. bassiana at the time of M. phaseolina inoculation
6. Soil inoculated with  M. anisopliae s.l. at the time of M. phaseolina inoculation
7. Soil inoculated with  B. bassiana 1 week after to M. phaseolina inoculation
8. Soil inoculated with  M. anisopliae s.l. 1 week after to M. phaseolina inoculation

Entomopathogenic fungi were applied as 1X10¹⁰ viable conidia in 100 ml of 0.01% Dyne-Amic (surfactant) solution distributed around the plant base.  To apply M. phaseolina, 5 grams of infested cornmeal-sand inoculum containing 2,500 CFU/gram was added to four 5 cm deep holes around the base of the plant.  Each treatment had six pots each planted with a single strawberry plant representing a replication.  Treatments were randomly arranged within each replication.  The study was repeated once a few days after the initiation of the first experiment.

Plant health was monitored starting from the first week after the M. phaseolina inoculation and continued for seven weeks.  Plant health was rated on a scale of 0 to 5 where 0=dead and 5=very healthy and the rest of the ratings in between depending on the extent of wilting.  Data from both experiments were combined and analyzed by ANOVA using Statistix software and significant means were separated using LSD test.  The influence of entomopathogenic fungal treatments applied at different times as well as the combined effect of different applications within each fungus were compared for seven weeks.  Ratings for some plants that were scorched from hot summer temperatures and died abruptly were removed from the analyses.

Results

Untreated control plants maintained good health throughout the observation period varying between the rating of 4.3 and 4.9.  In general, plant health declined considerably from the 5^th week after M. phaseolina inoculation.  Plant health appeared to be slightly better in plants treated with entomopathogenic fungi, but there was no statistically significant difference in any except one instance.  Plants treated with M. anisopliae one week prior to the application of M. phaseolina had a rating of 3.0 compared to 1.6 rating of plants inoculated with M. phaseolina alone.  

Plant health rating each week after inoculating with Macrophomina phaseolina.  Bars followed by the same letter within each week are not statistically different.

When data from different treatments for each entomopathogenic fungus were compared, both B. bassiana and M. anisopliae s.l. appeared to reduce the wilting, but the plant health rating was not significantly different from the M. phaseolina treatment alone.

This is the first report of the impact of entomopathogenic fungi on M. phaseolina with some promise.  Additional studies under more uniform environmental conditions and with more treatment options would shed more light on this approach of using entomopathogenic fungi against M. phaseolina.  The current study evaluated single application of the entomopathogenic fungi and we plan to conduct additional studies with multiple applications.

Acknowledgements: We thank Dr. Kelly Ivors (previously at Cal Poly San Luis Obispo) for the pathogen inoculum and Dr. Stefan Jaronski, USDA-ARS, Sidney, MT for multiplying the entomopathogenic fungal inocula.

References

Dara, S. K. and D. Peck.  2017. Evaluating beneficial microbe-based products for their impact on strawberry plant growth, health, and fruit yield.  UC ANR eJournal Strawberries and Vegetables. https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=25122

Dara, S. K. and D. Peck.  2018.  Evaluation of additive, soil amendment, and biostimulant products in Santa Maria strawberry.  CAPCA Adviser, 21(5): 44-50.

Dara, S. K., S.S.R. Dara, and S. S. Dara.  2017.  Impact of entomopathogenic fungi on the growth, development, and health of cabbage growing under water stress.  Amer. J. Plant Sci. 8: 1224-1233.  http://file.scirp.org/pdf/AJPS_2017051714172937.pdf

Dara, S. K., S. S. Dara, S.S.R. Dara, and T. Anderson.  2016.  First report of three entomopathogenic fungi offering protection against the plant pathogen, Fusarium oxysporum f.sp. vasinfectum.  UC ANR eJournal Strawberries and Vegetables. https://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=22199

Koike, S. T., G. T. Browne, and T. R. Gordon.  2013.  UC IPM pest management guidelines: Strawberry diseases.  UC ANR Publication 3468. http://ipm.ucanr.edu/PMG/r734101511.html

Partridge, D. 2003. Macrophomina phaseolina.  PP728 Pathogen Profiles, Department of Plant Pathology, North Carolina State University.  https://projects.ncsu.edu/cals/course/pp728/Macrophomina/macrophominia_phaseolinia.HTM

Vasebi, Y., N. Safaie, and A. Alizadeh.  2013.  Biological control of soybean charcoal root rot disease using bacterial and fungal antagonists in vitro and greenhouse condition.  J. Crop Prot. 2(2): 139-150.

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