Soil application of the entomopathogenic fungus Metarhizium brunneum protects strawberry plants from spider mite damage
Entomopathogenic fungus Beauveria bassiana is known to endophytically colonize various plants and provide protection against arthropod pests. Information of such endophytic interaction of another entomopathogenic fungus Metarhizium brunneum (=M. anisopliae) is limited.
A greenhouse study was conducted in 2010 to evaluate the endophytic potential of B. bassiana (commercial isolate GHA and a California isolate SfBb1) and M. brunneum (commercial isolate F52 and a California isolate GmMa1). Strawberry plants were grown in pots and fungal inocula were applied to the potting medium, vermiculite. When roots and aerial parts were periodically sampled, surface sterilized, and plated on selective media, B. bassiana grew from roots, petioles, pedicels, leaf lamina, sepals, and calyxes whereas M. brunneum was never detected from those tissues. It was initially thought that M. brunneum did not colonize strawberry plants.
However, there was an accidental infestation of twospotted spider mite, Tetranychus urticae on strawberry plants meant for another repetition of the endophyte study with M. brunneum isolates. Among those plants, 32 were treated with M. brunneum isolates and 20 were untreated control plants. Treatments were administered by applying 100 ml of conidial suspension at 1X10^10 conidia/ml concentration around the base of each potted plant. Each isolate had 16 strawberry plants. Mite counts were not taken as the plants were initially intended for endophyte evaluation and leaves could not be destructively sampled. But the proportion of plants damaged by mite infestations were recorded 10 and 14 days after fungal inoculation.
Plants treated with M. brunneum isolates appeared to withstand spider mite infestations better than untreated controls. Since M. brunneum could not be detected in the plant tissue in the previous attempt, it was not clear at that time how the fungus helped strawberry plants to withstand mite damage.
A recent study using scanning electronic microcopy showed that M. brunneum endophytically colonized cowpea plants. It is possible that M. brunneum colonized strawberry plants, but could not be detected using selective medium technique. Another study demonstrated that B. bassiana and M. brunneum promoted the growth of cabbage plants and improved the biomass. In the current study, M. brunneum probably improved the moisture absorption in strawberry plants through mycorrhizal interaction and helped withstand the spider mite infestations which are usually worse in plants under water stress. Fungal toxins in strawberry plants might have also impacted spider mites in a manner similar to the effect of endophytic B. bassiana on green peach aphid, Myzus persicae, in a different study. Observations from the current study indicate the potential of M. brunneum as an endophyte in protecting plants from arthropod damage. Additional studies are required to further investigate this interaction.
Acknowledgment: Thanks to Dale Spurgeon, USDA-ARS for providing laboratory and greenhouse resources for this study.
Dara, S. K. and S. R. Dara. 2015. Entomopathogenic fungus Beauveria bassiana endophytically colonizes strawberry plants. UCANR eNewsletter Strawberries and Vegetables, February 17, 2015.
Dara, S. K., S. S. Dara, and S. S. Dara. 2014. Entomopathogenic fungi as plant growth enhancers. 47th Annual Meeting of the Society for Invertebrate Pathology and International Congress on Invertebrate Pathology and Microbial Control, August 3-7, Mainz, Germany, pp. 103-104.
Golo, P. S., W. Arruda, F. R. S. Paixão, F. M. Alves, E.K.K. Fernandes, D. W. Roberts, and V.R.E.P. Bittencourt. 2014. Interactions between cowpea plants vs. Metarhizium spp. entomopathogenic fungi. 47th Annual Meeting of the Society for Invertebrate Pathology and International Congress on Invertebrate Pathology and Microbial Control, August 3-7, Mainz, Germany, pp. 104.
Vega, F. E., F. Posada, M. C. Aime, M. Pava-Ripoll, F. Infante, and S. A. Rehner. 2008. Entomopathogenic fungal endophytes. Biol. Con. 46:72-82.
Entomopathogen Beauveria bassiana is a soilborne fungus which is commercially available for pest management in organic and conventional agriculture. Although numerous studies demonstrated the interaction of B. bassiana with various arthropod hosts as a pathogen, information on its interaction with plants is limited. Some recent studies investigated the endophytic (growing inside the plant) interaction of entomopathogenic fungi with different species of plants in an effort to understand the impact on arthropods feeding on the plants and antagonistic effect on plant pathogens. When an entomopathogen is present in a plant as an endophyte, it may not cause infection in its arthropod host, but can affect its growth and development through (fungal) toxins. This interaction could be utilized to improve pest control efficacy and improve plant health.
To evaluate the ability of B. bassiana to endophytically colonize strawberry plants, two greenhouse studies were conducted in 2010 using a commercial isolate (GHA) and a California isolate (SfBb1). The first study examined three methods of inoculating strawberry plants where dry conidia of B. bassiana were mixed with potting medium (1X10^7 conidia/gram of vermiculite), strawberry roots were dipped in conidial suspension (1X10^7 conidia/ml) prior to planting, or 100 ml of conidial suspension (1X10^7 conidia/ml) was applied at the base the plant. Care was taken to prevent the contamination of aerial parts of plants with fungal inoculation. Each treatment had four potted plants and a set of untreated plants was used as control. Root, petiole or pedicel, and leaf lamina or sepal or calyx samples were collected 1, 3, and 6 weeks after inoculation to test for the presence of B. bassiana. Plant material was plated a selective culture medium after surface sterilization with bleach solution. Fungal growth from the plant tissue was microscopically examined and identified. Beauveria bassiana emerged from all plant tissues – roots underground to all aboveground parts – throughout the observation period. Among the inoculation methods, root dip and application of conidial suspension caused 52 and 44% of tissue colonization, respectively, followed by 4% colonization from mixing dry conidia.
The second study was conducted to evaluate colonization of B. bassiana at 1X10^9, 1X10^10, and 1X10^11 conidia/ml concentrations. Application of conidial suspension was chosen as it was the easiest means of inoculation and also practical to administer through drip irrigation system in the commercial fields. Treatments were administered by applying 100 ml of respective concentrations of conidial suspensions around the plant base. Plant tissues were sampled 1, 3, 6, and 9 weeks after inoculation using the abovementioned protocol.
Data were subjected to statistical analyses and significant means were separated using Tukey's HSD test.
Both commercial and California isolates colonized all sampled strawberry plant parts for up to 9 weeks after inoculation (Fig. 1). Due to the limited number of plants used in the study, sampling could not be continued beyond 9 weeks.
Fig. 1. Proportion of various plant parts endophytically colonized by commercial (GHA) and California (SfBb1) isolates of B. bassiana at 1, 3, 6, and 9 weeks after inoculation (WAI).
When concentrations were compared, fungal colonization of plants was the highest at 1X10^11 conidia/ml only for the commercial isolate (Table 1). There was no significant difference among conidial concentrations for the California isolate. In general, colonization was first noticed in roots and then the fungus moved up to the aerial parts. This trend was more evident for the commercial isolate with significant differences at 1X10^10 conidia/ml. Although not significant, it appeared that the commercial colonized strawberry plants more than the California isolate.
Table 1. Proportion of different strawberry plant parts endophytically colonized by commercial (GHA) and California (SfBb1) isolates of B. bassiana at various conidial concentrations.
*Average colonization of all plant parts for GHA isolate was significantly different at different concentrations P=0.03). Means followed by the same lowercase letter or no letter in the column were not significantly different.
**Colonization was significantly different among different plant parts at 1010 conidia/ml for GHA (P=0.01). Means followed by the same uppercase letter or no letter in the row were not significantly different.
These are the first studies to demonstrate that B. bassiana endophytically colonizes strawberry plants. The impact of endophytic B. bassiana on arthropod pests attacking strawberry plants was investigated in other studies.
Acknowledgment: Thanks to Dale Spurgeon, USDA-ARS for providing laboratory and greenhouse resources for these studies./span>
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The Food Safety Modernization Act
Comments about these revised rules are due to the FDA by December 15, 2014. If you haven't already done so, I encourage you to review the key revisions summarized on the FDA FSMA website (http://www.fda.gov/food/guidanceregulation/fsma/default.htm), as well as my comments below regarding some of the key revisions. You might also want to look at the National Sustainable Agriculture Coalition's excellent review of the FSMA: http://sustainableagriculture.net/fsma/; I reviewed their materials and borrowed some their language for this article.
You can submit your comments to the FDA at: http://www.regulations.gov/#!submitComment;D=FDA-2011-N-0921-0973 (for the Produce Rule) and http://www.regulations.gov/#!submitComment;D=FDA-2011-N-0920-1553 (for the Preventive Controls Rule).
Revised Definition of Covered Farms
However, the FSMA's $500,000 sales threshold (adjusted for inflation) for a farm to be eligible for modified requirements through a qualified exemption still relates to total farm revenues, rather than just produce sales. This cannot be changed because the text of the FSMA legislation specifically refers to “all food sales” A farm is eligible for modified requirements if it:
- has less than $500,000 in annual gross sales (adjusted for inflation) of all food products (includes commodities, hay, dairy, livestock as well as produce) over a previous three-year period AND
- sells the majority of the food directly to “qualified end-users”--consumers, restaurant and retail food establishment (e.g., a grocery store) that is located in the same state as the farm or not more than 275 miles from the farm.
Complying with the modified requirements means that a farm only needs to:
- Provide the name and complete address of the farm where the produce was grown on either a food packaging label or on a sign at the point of purchase;
- Comply with the “compliance and enforcement requirements” of the Produce Rule; and
- Be subject to the provisions regarding the withdrawal of your status as a partially covered (“qualified exempt”) operation; FDA can revoke your “qualified exempt” status in certain circumstances.
Broadened Definition of a “Farm”
Under the revised FSMA provisions, a farm that packs or holds raw agricultural products grown on another farm under a different ownership no longer has to register as a “food facility”. The original rule would have required a farm that aggregates produce from multiple farms for a CSA program to meet the Preventive Controls requirements. The revised rules will allow the farm to comply only with the Produce Rule. The “farm” may also:
- Pack or hold raw agricultural products;
- Manufacture or process food for on-farm consumption only;
- Dry/dehydrate raw agricultural products, as long as there is no additional processing; and/or label and package raw agricultural products as long as there is no additional processing.
Please be aware that the FSMA's terminology is very nuanced. Chopping or slicing fresh produce for sale – like carrots or apples – is considered to be processing, which means that you operate a “facility.” (Certain harvesting activities like trimming outer leaves of produce, or removing stems or husks, are not considered processing.). Washing is considered part of harvesting when done in the field. But, if it is done during the production of fresh-cut produce, for example, it is considered manufacturing or processing. Labeling and packaging are considered manufacturing activities unless you are labeling or packaging a raw agricultural product and are not doing any additional manufacturing or processing to the product. If there is no additional manufacturing or processing, labeling and packaging are considered farm activities and do not trigger the facility definition.
Revised Water Quality Standard and Testing Are More Flexible
For farms that have to test their water, the FDA is proposing three numerical standards below for testing.
- No detectible E. coli present per 100 ml of water: This standard would apply to water used for an activity during and after harvest, water used to make agricultural teas, and water used in sprout irrigation. The quality of untreated surface water used for these purposes must be tested from each source of the water “with an adequate frequency to provide reasonable assurances that the water meets the required standard”. You must have adequate scientific data or information to support your testing frequency.
- Farms using untreated groundwater for purposes that trigger a testing requirement will now have to test their water supply a maximum of 5 times in the first year (4 per year/growing season plus one test per year) rather than testing on a quarterly basis as originally proposed. Their untreated groundwater used to irrigate in a manner that directly contacts the harvestable portion of the crop will have to meet the following standard: a geometric mean of no more than 126 colony forming units (CFUs) per 100 ml.
- Testing of untreated surface water used for growing produce other than sprouts involving direct contact with the harvestable portion will require the collection of 20 samples over the first 2 years, followed by an annual minimum sampling of 5 per year, rather than monthly or weekly as previously required. The water will have to meet the following standard: a statistical threshold value (STV) of 410 CFUs generic E. coli per 100 ml for a single water sample, and a geometric mean of no more than 126 CFU per 100 ml. If your water testing shows that you exceed these values, you can still use your water, as long as you apply an appropriate time interval between the end of irrigation and harvest as determined by calculating the “microbial die-off”.
Clarification of Provisions on Wild Animals
Manure Application Interval Will Be Studied Further
The FDA had previously proposed a nine-month minimum time interval between the application of untreated soil amendments of animal origin (including raw manure) and harvesting. This requirement conflicted directly with the USDA National Organic Program's standards, which require a 120-day interval between the application of raw manure for crops in contact with the soil and 90 days for crops not in contact with the soil. The FDA now proposed to conduct a risk assessment and extensive research to strengthen scientific support for any future proposal. Additionally, the FDA is proposing to eliminate its previously proposed 45-day minimum application interval for compost.
Significant Compliance Costs Remain for Small-scale farms
The provisions reviewed above all were improvements over the original FSMA Produce Rule. The revised rules reduced the estimated number of farms in the United States covered by the FSMA by 4,708, of which 2,885 are “very small”. However, some of the provisions still impose disproportionately high compliance costs on smaller-scale farms, as indicated below in Table A. Smaller-scale farms typically have very constrained cash flows. The added expenses to comply with the FSMA makes their cash flows even tighter and reduces their already low level of profitability. If a "very small" farm loses over a fifth of its net cash farm income, this could have significant impacts on its sustainability. And these decreases do not include the one-time capital expenses that a farm may have to incur, such as to modify restrooms or handwashing facilities and to build fences!
The percentage decrease in net cash farm income attributable to the costs of complying with the FSMA declines as farm size increases. Clearly, there are economies of scale in complying with the FSMA. The FSMA includes delayed implementation of FSMA compliance for smaller-scale farms. Policymakers should also consider providing subsidies and/or no-interest loans for the capital expenditures smaller-scale farms need to make to comply with the FSMA.
Again, please consider submitting comments to the FDA about the proposed FSMA Produce Rule and the Preventive Controls Rule. You can post your comments using the links at the beginning of this article. You can review the FSMA's key revisions summarized on the FDA's FSMA website (http://www.fda.gov/food/guidanceregulation/fsma/default.htm). Also consider reading at least part of the National Sustainable Agriculture Coalition's excellent review of the FSMA.
Understanding the behavior of a pest is very important in developing appropriate control strategies. Information on feeding, host searching, migratory, and reproductive behavior of the invasive Bagrada bug is very limited in published literature. Since Bagrada bug is a fairly new pest in the United States, there is a lot to learn and understand about this pest. Here is a summary of observations about its feeding and reproductive behavior.
Bagrada bugs are primarily attracted to cruciferous crops. However, the number of host species this pest feeds on or passing through is increasing as it spreads to different parts of California. In addition to various wild and cultivated cruciferous plants, Bagrada bugs have been reported to cause damage to carrots, corn, peppers, potatoes, tomatoes, and sunflower. In an earlier choice study where different host plants were offered, neither adults nor nymphs chose tomatoes when alyssum, broccoli, green bean, and wild mustard were among the choices (Dara and Dara, 2013). However, feedback from some growers this year indicated feeding damage to tomatoes (Dara 2014). Although damage was not confirmed, some growers and homeowners reported finding Bagrada bugs on citrus, fig, grape, and strawberry.
Condition of the plants
During a visit to a home garden a couple of years ago, I noticed several Bagrada bugs on dried branches of wild mustard, although different cruciferous vegetable plants were in the proximity. Considering the ability of Bagrada bugs to move around easily, this observation suggests their preference for certain plant conditions. In a recent visit to a 4-week old broccoli field, Bagrada bugs and their damage was noticed only on small and weak plants. Heavy winds a few weeks earlier affected some plants which were significantly smaller than the rest of the plants and were breaking at the base with a slight touch. Similarly, in my lab colony, several bugs are frequently seen on relatively drier plant material although fresh plant material is also present. All these observations suggest that the concentration of plant juices could be influencing Bagrada bugs choice within a specific host. This could mean that maintaining good health of the plants through optimal irrigation and nutrient management is important to avoid weaker plants that could attract Bagrada bugs.
Bagrada bugs are known to hide in the cracks of top soil during cooler parts of the day. Even during warmer parts of the day, some bugs were seen in the soil. This behavior could be exploited by the use of entomopathogens such as Beauveria bassiana and Metarhizium brunneum, which are soilborne fungi. Applied through drip irrigation or as a foliar spray, these fungi can be introduced into the Bagrada bug habitat. Natural behavior of the Bagrada bug to dwell in the soil increases its chances of exposure to fungal inoculum. Although solar radiation might inactivate fungal spores on exposed plant surfaces, being soilborne fungi, these pathogens can persist in the soil for longer periods. Preliminary laboratory assays already demonstrated the potential of these fungal pathogens (Dara 2013).
Based on laboratory observations, Taylor and Bundy (2013) indicated that Bagrada bugs preferred dry soil compared to moist soil to deposit eggs. While this might be the case when Bagrada bugs feed on wild hosts in uncultivated areas, cultivated crops are frequently irrigated and how the soil moisture influences their oviposition behavior in the field conditions is not clear. Earlier literature indicated that eggs are also deposited on various plant parts. Whether eggs are deposited on the plant or in the soil, entomopathogenic fungi could still be important to cause mortality in newly emerged nymphs that might walk on fungal inoculum. If Bagrada bugs overwinter as eggs in the soil, cultivation can be a tool to reduce their numbers. Some entomopathogenic fungi cause egg mortality in addition to infecting mobile stages.
Nature and Numbers
Bagrada bugs have a wide host range and some of their preferred hosts are spread across large areas as wild plants. When these plants dry out, they migrate to crop plants in significant numbers. This is probably why control with pesticide applications alone or using trap crops can be challenging. Some community and home gardeners who tried to use trap crops or traps with alyssum, were able to find large numbers in those crops or traps, but even larger numbers continued to move to crop plants. For a pest like Bagrada bug, exploiting natural enemies appears to be a crucial management tool. Arrangements for foreign exploration of natural enemies are underway.
Dara, S. K. 2013. Bagrada bug update: bioassays and a short video.
Dara, S. K. 2014. Current status of the invasive Bagrada bug in California: geographic distribution and affected host plants.
Dara, S. K. and S. S. Dara. 2013. Bagrada bug host preference: crucifers and green beans.
Taylor, M. and C. S. Bundy. 2013. The life history and seasonal dynamics of Bagrada hilaris in New Mexico. Annual meetings of the Entomological Society of America, Austin, TX.