Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

Hi.My name is Ush Parsa and I'm an entomologist at SEA at the International Center for Tropical Agriculture. I'm ViiV Artiz, also from the International Center for Tropical Agriculture, and I am Fernando Vega from the United States Department of Agriculture. The overall objective of this video is to demonstrate how to facilitate and verify the establishment of a fungal into pathogen.

As an endophyte, It is common in nature to find an insect or a mite infected with a pathogenic fungus, and there's a long history of using these fungal ando pathogens as biological pesticides. More recently, fungal ando pathogens have been occasionally found infecting plants, but without producing any apparent symptoms of their presence. This discovery signals their potential use as protective endophytes against agricultural pests.

To explore this potential, it is first essential to develop an inoculation method that reliably establishes a fungal pathogen as an endophyte in a target crop. B Bassiana is one of the best studied fungal pathogens, and it is readily available as a commercial by a pesticide. This fungus has been detected as an endophyte in many plant species, including bananas, coffee and mace.

Recent evidence suggested in the B bassiana has the potential to protect plants not only from arthropod pests, but also from some plant pathogens. The mechanisms explaining this broad spectrum protection are poorly understood. Still in the tic valve area, may hold promise for crops lacking genetic resistance to a pest or a pathogen, but for crops that typically suffer from the simultaneous attack of multiple enemies, the common being fas vulgaris is a prime example.

It can be affected by more than 400 pests and 200 pathogens whose attack is thought to be the most limiting bean production factor across regions. For this reason, we feel the common be may be a good candidate crop to examine the protected potential of Endo bbar. As a first step in this direction, Arvid demonstrates the efficacy of foliar sprays and soil drenches as inoculation methods to establish Bana as an endophyte.

In the common bean seed from the bean called TOMA are surface sterilized by a two minute immersion in 0.5%sodium hypochlorite, followed by a two minute immersion in 70%ethanol and three rinses in sterile distilled water to theorization success. A hundred microliter sample of the rinsing water is plated on a 100 millimeter Petri plate with PDA media and incubated in the dark for 10 days at 25 degrees Celsius. The experiment should be aborted and restarted if growth is seen in these controls.

Seeds are sewn in groups of three in pots 11.5 centimeters wide, containing a vapor sterilized mixture of soil and sand at a two to one ratio. Plants are grown under controlled conditions at 25 degrees Celsius, 50%relative humidity and a 12 hour photo period with a light intensity of 9, 200 lux a week after germination. The plants are thin to one per pot to ensure vigor and eliminate competition.

They're irrigated as needed every two to three days and fertilize 10 and 20 days after planting with a six grand per liter water solution of NPK fertilizer. To ensure genetic uniformity of our fungi, isolates should be grown from single Canadian cultures. If the source of the fungus is an insect with mycosis, we take the specimen to the laminar flow hood and use an inoculating loop to transfer a small sample of DYS sporulating fungus to PDA media.

Subsequent transfers to new media plates may be necessary to remove contaminants once a target fungus grows and spates in a clean culture. The small sample of edia suspended in one milliliter of a 0.1%water solution of Triton X 100 and vortex tritton Exus is surfactant that reduces water surface tension and allows kedia to disperse. A 100 microliter aliquot of this suspension is spread on a 100 millimeter Petri plate containing a thin layer of 2.5%noble acre, which will allow for visualization of kedia on their stereoscope.

The plate is incubated at room temperature for 24 hours in the dark, after which a single germinating pen medium is transferred into the media plate with PDA and allowed to grow for three to four weeks until it covers the entire media plate and spates. This method provides a pure culture of the fungus for subsequent experiments. For this demonstration, we use a pure culture of Bavaria Bassiana, a strain GH HA, obtained from the commercial product Microt.

The inoculum is prepared under sterile conditions. In a laminar flow hood, we use a sterile spatula to scrape approximately one gram of the fungal growth from the surface of the medium and suspended in 10 milliliters of sterile 0.1%Triton X.The suspension is vortex for one minute and filtered through sterile cheesecloth to separate the canadia from the Hy-Vee. This step yields a stuck suspension that needs to be adjusted to a final concentration of 10 to the eight kedia per milliliter for inoculations.

First, the concentration of the stock is estimated in a serial dilution that facilitates counting kedia. In a Hema Cytometer, 100 microliters of the stock are diluted in 900 microliters of 0.1%Triton X and vortex resulting in a one in 10 dilution. The procedure is repeated serially three additional times to obtain a one in 10, 000 dilution.

Kedia will be present in extremely high numbers in the first dilution and in low numbers that can be easily counted in the last dilution. Each of the two chambers of the Hema cytometer is loaded with 10 microliters of the one in 10, 000 dilution and placed under a microscope to count kedia. The ruled area of the Hema Cytometer consists of nine large squares each holding 100 nanoliters or 0.0001 milliliters accordingly.

To estimate the concentration of ED in the sample, we count and average the ED in the four large corner squares of each chamber and divide that average by 0.0001. Multiplying this concentration by our dilution factor 10, 000 gives the concentration of the stock now to calculate the final volume to which a stock must be adjusted. To obtain the 10 38 edia per milliliter inoculum, we apply the formula.

Final volume equals stock volume times stock concentration over final concentration. In our demonstration, the stock needs to be adjusted to a final volume of 195.5 milliliters to yield our desired concentration. Before using the inoculum, it is important to conduct a germination test assessing canal viability.

For this test, we place 100 microliters of the one in 10, 000 dilution on a 100 millimeter Petri plate with 2.5%noble egg or media. The sample is spread with a sterile glass rod into the medium surface and incubated in the dark at 25 degrees Celsius for 24 hours. Viability assessed under the microscope by estimating the percentage of kedia with growing germ tube.

For three random samples of 100 Canadian, the Canadian suspension can be used for inoculations only if average germination is above 90%In this demonstration, we find an average germination of 97%We will demonstrate two inoculation methods, foliar sprays and soil drenches both applied when plants reach their first true leaf stage. Approximately 14 days after planting, the foliar spray method is performed with a manual atomizer directing the cial suspension to the al or upper surface of leaves until they're saturated. The top of each pot is covered with aluminum foil to avoid conal runoff to the soil After spraying, each plant is covered with a plastic bag for 24 hours to maintain high levels of humidity facilitating fungal invasion.

The soil drench inoculation method is conducted at soil water capacity. Approximately 24 hours after watering plants to soil saturation, 10 milliliters of a cial suspension are applied to the surface of its soil. At the base of the plant controlled receive a steril 0.1%Tritton X applied in the same way as in their corresponding treatments.

Plants are returned to growth chambers and arranged in randomized complete block design with three replicates per treatment. We evaluated the the establishment two weeks after inoculations one block at a time. The plants are carefully harvested and individually washed in running tap water from each plant.

We sample two leaflets, two pieces of root and two pieces of stem leaflets are randomly selected from the first true leaf of the plant and trimmed to squares stem. Samples of roughly three centimeters long are obtained from the middle of the plant and from near the soil surface. Tap root samples of a similar length are obtained from the middle of the root and from one centimeter behind the root tip.

Each sample is individually surface sterilized in a sterile hood in 0.5%sodium hypochlorite for two minutes, followed by 70%ethanol for two minutes and rinse three times with sterile distilled water before letting it dry. In sterile towel paper, the sterilized sample is then carefully dissected to discard its outer edge where endophytes might have been eliminated due to contact with disinfectants ants. The trim sample is then cut into smaller sections averaging six by six millimeters for leaves and six millimeters long for stems and roots.

The sections are plated on 60 millimeter Petri plates containing PDA supplemented with the antibiotics tetracycline as streptomycin and penicillin at two milligrams per liter each. Finally, the plates are sealed with paraffin and incubated in the dark at 25 degrees Celsius. The rinse water is changed after processing each block, but before a 100 microliter sample is plated on a 100 millimeter Petri plate, PDA media and incubated for 10 days at 25 degrees Celsius to determine if the serialization was successful.

If any growth is seen in this control, the block is discarded and not considered for analysis. The plates are inspected every two to three days for 20 days to observe and record fungal growth. Colonized plant sections are excised and transferred individually to new media plates to avoid contaminating neighboring sections.

B bassiana growth is recorded based on characteristic white dense mycelia becoming cream to pale yellow at the edge. When in doubt, the sample is mounted in a drop of water on a microscope light and examined for global scania and zigzag shape kedia force characteristic of the species on the same day colonization is evaluated. We also estimate the impact of our treatments on plant growth.

We first measure plant height from the base of the plant to the top of the growing point. Then we carefully uproot and wash plants in tap water and let them dry at 45 degrees Celsius for three days to measure their dry weight. For this demonstration, we conducted the full experiment twice to obtain representative results.

Both inoculation methods resulted in the physicalization by Bana in over 80%of treated plants. However, the extent of colonization dependent on the plant part evaluated and the inoculation method used. Leads responded best.

Spray inoculations roots, on the other hand only responded to drench inoculations. Finally stems responded similarly to both inoculation methods. Bana was not detected in any of the controlled plant sections independent of the treatment endophytes other than Bana grouped from 15%of the evaluated plant sections, but they were dissected out from media plates before they could invade neighboring sections and influence our results.

Treatment and control plants were visibly indistinguishable two weeks after inoculations, and no significant differences were detected in their dry weight and in their height. Many factors can influence a specific outcome of an experiment to establishing endo pathogen as an end effect. As this video demonstrates, the inoculation method is one of them biological factors to experiment with include crop species or cultivar selected and the choice of the OMA pathogen species or isolate other factors to consider manipulating include the concentration of the inoculum, the age of the plant during inoculations and the plant's growing conditions.

The ultimate goal for these experiments should be to develop an efficient treatment that provides durable systemic resistance against INE or disease. When you're ready to test for Endof fight mediated resistance, we recommend you watch a video characterizing Herbal Resistance Mechanisms also published at the Journal of Visualized Experiments. Please be aware that endophytes are very common in plants and they're likely to influence your ability to persistently establish and detect a target fungal to pathogen as an endo fight.

We hope we find this demonstration useful. Good luck with your experiments.