Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
Plant Pathology
Non Technical Summary
The world has lost a third of all arable land in the last 40 years due to erosion and pollution damage, yet we remain on track to exceed global population of nearly 10 billion people and double our food production needs by 2050. Greenhouse agriculture and controlled environment agriculture (CEA) are growing rapidly as viable solutions to this problem. Greenhouse/CEA has some fundamental benefits in addressing societal needs including i) year round growing mitigating the effects of weather, ii) flexibility of farm locations reducing shipping time and cost iii) potential health benefits from locating farms near the 24 million Americans currently living in food deserts without access to nutritious fresh produce, iv) water use efficiency, and v) increased food safety and traceability from growing in controlled soilless systems which inherently limits exposure to soil borne pathogens. The North America greenhouse market is growing rapidly to meet society's need for fresher, locally grown produce. Currently >75% of fresh-market tomatoes and nearly 100% of cucumbers sold in supermarkets are produced in greenhouse operations. By design, greenhouse systems offer great potential for more efficient use of water, fertilizer, and pesticides in food production. Most largescale greenhouse operations use soil-less growth systems (i.e., hydroponics) which can protect plants from drought and pests common to vegetable field production. However, re-use of water through nutrient recirculation systems offers a pathway for rapid proliferation of waterborne microorganisms. In the presence of high concentrations of nutrients, certain plant pathogens can rapidly grow and disseminate throughout the water system to contaminate an entire crop, leading to significant crop loss as well as substantial "down time" for disinfection before a new crop can be planted. Disease threats such as crazy root (caused by Agrobacterium rhizogenes) and root-rots caused by Pythium, Phytophthora, and Fusarium spp. are commonly observed in greenhouse systems.Current prevention of plant pathogens in hydroponic systems includes ultraviolet or gamma irradiation and/or chemical treatments. Use of beneficial microbes is of increasing interest because irradiation systems are expensive and require periodic maintenance, and chemical treatments have raised issues with phytotoxicity and development of pathogen resistance. Additionally, after planting crops in a hydroponic system, a microflora naturally develops, some of which may be plant pathogens but more commonly are non-pathogenic microorganisms. Researchers have found the natural microflora's beneficial effects not big enough to prevent disease outbreaks. Therefore, the addition of targeted beneficial microbes to the recirculating nutrient system may offer a practical means to help manage a healthy microbiome and prevent plant disease outbreaks in hydroponic systems. To this end, we are screening a library of different Pseudomonas strains developed in the Taylor laboratory for their ability to control plant pathogens that frequent hydroponic systems. Pseudomonas strains that inhibit plant pathogens under in vitro conditions will be tested in planta to determine if disease development can be inhibited in hydroponic systems.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
Pseudomonads (Pseudomonasspp.) are naturally occurring bacteria that are common to plants and can exhibit a range of crop-enhancing properties. Pseudomonads have been widely studied for their biocontrol activity against numerous plant pathogens (including, most notablyPythiumspp.andPhythopthoraspp.), are excellent root colonizers, and can sometimes exhibit plant growth promoting activity.These bacteria can produce a variety of bioactive compounds and proteins, including 2,4- diacetylphloroglucinol (DAPG), hydrogen cyanide (HCN), auxin, lipo-polysaccharides, chitinase, harpins and exoproteases. Specific to hydroponic systems, severalPseudomonasspp. have shown promise in research to increase yield and suppress root pathogens in tomatoes, peppers, lettuce, and cucumbers. The Taylor laboratory has sequenced and bioinformatically annotated over 50 different strains ofPseudomonasbacteria, originally isolated from a library of 12,000 microbial isolates purified from over 500 water, soil, and plant samples collected from around the United States. This strain collection has been screened for in vitro activity against bacterial (Agrobacterium tumefaciens,A. rhizogenes,A. vitis,Ralstonia solanacearum,Erwinia amylovora,andClavibactor michiganesis), fungal (Fusarium viguliformeandF. oxysporium,Rhizoctonia solani,Sclerotinia sclerotiorum,Colleotrichum acutatum,Thielaviopsis basicola, andBotrytis cinerea) and oomycete (Phythium cryptoirregulare,Phytophthora cryptogeaandPhytophthora sojae) plant pathogens mostly under in vitro conditions. Despite the significant body of research on the benefits ofPseudomonasspp. over the past 30 years, very little successful commercialization has occurred to allow growers to benefit from this knowledge. Only a few commercial products based onPseudomonasare available, none of which are registered for use in hydroponic systems.The initial focus is addressing an emerging disease of hydroponically grown tomatoes, peppers and cucumbers called Crazy Root disease (CR; caused by Agrobacterium rhizogenes). Plants infected by A. rhizogenes results in the formation of transgenic adventitious roots that form a large root-mat that robs the plant of nutrients normally bound for shoot and fruit production. The observed increase in root proliferation also leads to the pushing of the plant out of the hydroponic sleeve and to blockage of the nutrient channels leading to decreased water and nutrient flows of downstream plants. Furthermore, the large root masses produce ideal conditions for infection by other plant pathogens including Pythium spp., leading to a decrease in plant vigor and even death. In the 1990s, CR became a significant issue on tomatoes grown on rockwool slabs as an inorganic base media that allowed for efficient movement of hydroponic liquid. Crazy Root disease has now spread across Europe, Russia, parts of Asia, and now the United States. In Europe, the occurrence of CR disease in tomato has increased from 8% (pre-2011) to 25% (in 2012) and continues to grow. In the United States, most greenhouse operations have observed CR in their growing systems and CR is now considered one of the leading biotic stressors for greenhouse operations. One of the major issues with CR is that as little as one infected plant may be able to infect an entire greenhouse operation. Experiments indicate that CR on tomato reduces yield by 10-15% which can significantly impact a grower's return on investment. In addition to affecting plants and reducing yield, the growers at the end of the growing cycle must sterilize or throw away all infected pipes and troughs and sterilize the water delivery system leading to added expenses and operational downtime.To identify a potential biocontrol agent for CR, the Ohio State University's Pseudomonas collection of bacteria was screened in a top-agar zone of inhibition study using agrobacteria (A. rhizogenes strain K599). Of the 52 different Pseudomonas strains tested, 14 inhibited the growth of A. rhizogenes in in vitro experiments. These 14 strains were then tested in a plant assay on Kalanchoe (a succulent that is very susceptible to agrobacteria infection). After three weeks, disease incidence (the formation of roots at the infection site) was measured. Of the 14 plate-inhibitory strains, three strains were identified that reduced the incidence of disease formation in planta. Strains 93G8 (P. brassicacearum) and 1B1 (P. protegens) reduced transgenic adventitious root formation by 100% while strain 48G9 (P. chlororaphis) reduced it by 50%. These three strains come from different sub-groups within the Pseudomonas genus, thus increasing the probability that they exhibit different modes of action (also supported by limited overlap of known genes associated with biocontrol, data not shown). To further quantify the reduction in formation of CR, a separate in planta bioassay was developed using greenhouse grown soybeans. A previous assay used to produce transgenic roots was modified, using A. rhizogenes strain K599 containing a binary vector with T-DNA encoding for the green fluorescent protein (GFP). After four weeks, the ability of A. rhizogenes to induce transgenic adventitious root formation (determined from natural roots by the presence of GFP-expression roots) at the cut site on the soybean stem was determined by examining the roots that formed under a fluorescence microscope. The addition of Pseudomonas in general reduces the incidence of transgenic adventitious root formation, however, those strains identified as being A. rhizogenes-inhibitory showed an even larger reduction in transgenic adventitious root formation. Strains 93G8 and 1B1 reduced the incidence of transgenic adventitious roots by 95% as compared to the untreated (non-Pseudomonas containing plant growth media) control. Though the initial focus of this proposal is on the development of these three strains for CR control, we are also in the early stages of identifying Pseudomonas strains that inhibit other pathogens that frequent hydroponic systems. The Taylor laboratory is investigating the possibility of these and other Pseudomonas strains from the OSU Pseudomonas collection to control fungal/oomycete hydroponic-related diseases.Using target Pseudomonas spp. strains, the overall technical objectives will be to: 1) Evaluate efficacy in hydroponics trials on CR disease with both induced disease pressure and under typical operating conditions. 2) in vitro and in planta testing of Pseudomonas strains for their ability to inhibit fungal and/or oomycete diseases that frequent hydroponic systems. And 3), determine mode of action for some of these bioactive strains using mutagenesis and/or complementation.
Project Methods
Evaluate efficacy in hydroponics trials on CR disease with both induced disease pressure and under typical operating conditions.??Experiment 1: Pilot scale testing of strains. The Taylor laboratory has established a tomato-based hydroponic system within the Plant Pathology greenhouse facility in Wooster, OH. The sterilizable system allows direct application of microbes and pathogens under actual hydroponic conditions. Each tray will be pretreated with the test Pseudomonas strains (individually or in combination). Two-week-old tomato plants (var. Money Maker) grown in oasis cubes or soilless media will be placed in the individual rockwool cubes. Plants will then be treated with an adequate dose of A. rhizogenes to ensure disease at 6, 12, 24, 48 and 72 hours after treatment. Plants will be examined for the presence of CR and the efficacy of treatment determined. Several controls will be utilized, including mock inoculated, non-treated, and a non-disease-causing variant (i.e. cured) strain of A. rhizogenes.Experiment 2: Combination testing of strains for synergistic effects. To further examine the potential of Pseudomonas to inhibit CR disease, the Taylor laboratory will also test combinations of Pseudomonas to determine if there is any improvement in pathogen control above single strain application. We are uncertain at this time whether combinations of beneficial bacteria will lead to additive, synergistic or reduced activity in a hydroponic system. Double and triple combinations will be tested to determine if there is any change in biocontrol activity.Experiment 3: Yield testing. Yield testing will be conducted using Pseudomonas grown in the absence of any disease pressure. Yield testing will be conducted using Bato BucketTM system used for growing vine tomatoes for fruit production. The Taylor laboratory has nearly 70 of these set up in the Plant Pathology greenhouse and is currently using them to measure yield reduction by A. rhizogenes (ongoing experiments; data still being collected). Tomato plants (var. Rebelski -F1) will be germinated in rock wool cubes and transferred to the individual Bato Buckets and inoculated with Pseudomonas. We will initially apply Pseudomonas once every month to the root system of the plant. Plants will be measured for shoot height and leaf surface area. Plants will be trellised and allowed to produce fruit for three months. Ripe fruit will be harvested throughout the experiment and weighed. At the end of three months, all fruit will be harvested and weighed.In vitro and in planta testing of Pseudomonas strains for their ability to inhibit fungal and/or oomycete diseases that frequent hydroponic systems.Experiment 1: Pilot scale testing of strains. The Taylor laboratory has established a spinach-based deep well hydroponic system within the Plant Pathology greenhouse facility in Wooster, OH. The sterilizable system allows direct application of microbes and pathogens under actual hydroponic conditions. Each tub/bucket will be pretreated with Pseudomonas strains (individually or in combination). Two-week-old spinach plants (var. Bloomsdale) grown in oasis cubes or soilless media will be placed in the individual plant baskets and place on Styrofoam floats. Plants will then be treated with an adequate dose of Pythium aphanidermatum to ensure disease at 48 hours after Pseudomonas treatment. Plants will be examined for the presence of root rot (browning of the roots) and the efficacy of treatment determined over the next three weeks. Several controls will be utilized, including mock inoculated, non-treated, and a surfactant with known Pythium killing abilities.Experiment 2: Combination testing of strains for synergistic effects. To further examine the potential of Pseudomonas to inhibit root-rot disease, the Taylor laboratory will also test combinations of Pseudomonas to determine if there is any improvement in pathogen control above single strain application. We are uncertain at this time whether combinations of beneficial bacteria will lead to additive, synergistic or reduced activity in a hydroponic system. Double and triple combinations will be tested to determine if there is any change in biocontrol activity.Experiment 3: Yield testing. Yield testing will be conducted using Pseudomonas in the absence of any disease pressure. Yield testing will be conducted using the deep-well tub/bucket system previously described. The Taylor laboratory has 64 of these set up in the Plant Pathology greenhouse and is currently using them to measure yield reduction by p. aphanideratum (ongoing experiments; data still being collected). Spinach plants (var. Bloomsdale) will be germinated in rock wool cubes, dipped into a Pseudomonas solution and then transferred to the individual plant baskets on Styrofoam floats. We will initially apply Pseudomonas once every month to the root system of the plant. Plants will be measured for shoot height and leaf surface area.Determine mode-of-action for these individual strains using mutagenesis and/or complementation. Generation of Pseudomonas mutant libraries. We will use random mutagenesis to produce knockouts mutants in Pseudomonas. Pseudomonas will be transformed with the Epicentre's EZ::TNTM TransposomeTM kit and kanamycin-resistant colonies will be picked into a 96-deep-well plant containing Kings-B medium and grown overnight at 30oC. In vitro bioassays using A. rhizogenes and/or P. aphanidermatum will be used to identify mutants that have lost their biocontrol activity. Loss-of-function mutants will be screened to identify what genes have been knocked out. Complementation experiments will be used generated by reintroducing the gene back into mutants using compatible plasmids to determine if biocontrol activity is restored. Previous results done in the lab on Pseudomonas loss-of-lethality against the nematode, Caenorhabditis elegans showed an average knockout rate of 0.88%. We anticipate that screening 1,000 colonies (~10 plates) should produce about eightmutants that are defective in biocontrol activity.