ABSTRACT
TAYLOR, MARY CLAIRE GARRISON. Mycophagous Soil Fauna for Biological Control of Soilborne Pathogenic Fungi in Greenhouse and Transplant Crops. (Under the direction of Shuijin Hu and H. David Shew).
The objective of this project was to investigate the feasibility of using dairy cow compost as a soil amendment in conjunction with nematodes, collembolans, and mites as an alternative to chemical control for soilborne fungal pathogens and their diseases in greenhouse and transplant crop production systems. Three model systems were used; 1)
Tomato attacked by Pythium ultimum, 2) Vinca and Impatiens attacked by Rhizoctonia solani, and 3) Bell pepper attacked by Phytophthora capsici. Five experiments total were conducted using three types of faunal treatments; 1) Nematode Aphelenchus avenae, 2)
Collembolan (unidentified), and 3) Mite (unidentified) and three rates of dairy cow compost;
1) 4% by volume, 2) 8% by volume, and 3) 16% by volume. The fungal-feeding nematode
Aphelenchus avenae can effectively suppress seedling damping-off caused by Pythium ultimum, Rhizoctonia solani, and Phytophthora capsici to a level equivalent to the standard fungicide treatment. Compost rate affects the disease suppression efficiency of the faunal treatments likely through impacting the mesofauna’s survival and growth in the seedling rhizosphere. Disease suppression capacity resulting from mesofauna may be extended to the field. Mycophagous Soil Fauna for Biological Control of Soilborne Pathogenic Fungi in Greenhouse and Transplant Crops
by Mary Claire Garrison Taylor
A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science
Plant Pathology
Raleigh, North Carolina
2010
APPROVED BY:
______Shuijin Hu H. David Shew Committee Co-Chair Committee Co-Chair
______D. Michael Benson Frank J. Louws
DEDICATION
To Troy Ashton Taylor
ii BIOGRAPHY
The author was born in Virginia but spent most of her childhood in North Carolina.
She was a class of 2006 Park Scholar at N.C. State and received her B.S. in Microbiology in
December 2007. She and her husband met as graduate students in Plant Pathology at N.C.
State.
iii ACKNOWLEDGMENTS
I would like to thank my co-advisors Dr. Shuijin Hu and Dr. H. David Shew, as well as my committee members Dr. D. Michael Benson and Dr. Frank J. Louws for all their time and hard work on my behalf during my time as a graduate student. I would also like to thank the other members of the Soil Ecology lab, Cong Tu, Lei Chen, and Liang Wang for their support.
Dr. Barbara Shew hired me in my sophomore year to work as an undergraduate research assistant to work in the “peanut lab” so I have her to thank for introducing me to the world of Plant Pathology. Her graduate student, now Dr. Damon L. Smith, and research technician Joyce Hollowell, taught me more about peanut diseases than I ever really wanted to know! They were both also key in my decision to apply to graduate school in the NCSU
Department of Plant Pathology and to them I also owe great thanks.
The biggest thank you is of course owed to my parents. I can’t possibly mention everything I have to thank them for, because the magnitude is just too great.
I love you Mom and Dad.
iv TABLE OF CONTENTS
LIST OF TABLES ……………………………………………………………… vii LIST OF FIGURES ……………………………………………………………... viii CHAPTER 1. SOIL BIOLOGICAL INTERACTIONS AND BIOLOGICAL CONTROL OF FUNGAL PATHOGENS IN GREENHOUSE CROP AND FIELD TRANSPLANT PRODUCTION SYSTEMS …………………...... 1 1.1 Greenhouse crop and field transplant production ……………………. 1 1.1.1 The scope of greenhouse and transplant crop production ………..... 1 1.1.2 Soilborne fungi in greenhouse systems ……………………………. 2 1.2 Soilborne fungal pathogens of plants and their biological control…… 3 1.2.1 Soilborne funal pathogens………………………………………….. 3 1.2.2 Biocontrol of soilborne fungal pathogens…………………………... 7 1.3 Soil food web interactions and biocontrol …………………………… 17 1.3.1 Soil mesofauna and soil food web interactions ……………………. 17 1.3.2 Soil mesofauna as biocontrol agents ………………………………. 23 1.4 Integrated biological control of fungal pathogens and diseases in greenhouse and transplant crops: Opportunities and challenges …….. 27 1.4.1 Fungal-feeding animals and soil amendments …………………….. 27 1.4.2 Biological control in greenhouse and transplant crops …………….. 28 1.4.3 Integrated biological control ……………………………………….. 29 1.4.4 The overall goal and specific objectives of this research ………….. 30 References ……………………………………………………………….. 31 CHAPTER 2. BIOLOGICAL CONTROL OF DAMPING-OFF DISEASES OF GREENHOUSE VEGETABLE CROPS AND BEDDING PLANTS …………... 42 2.1 Introduction …………………………………………………………… 42 2.1.1 Host-pathogen systems …………………………………………….. 43 2.1.2 The specific objective……………………………………………….. 45 2.2 Materials and methods …………………………………………...... 45 2.2.1 Preparation of inoculum of nematode Aphelenchus avenae and Pythium ultimum, and germination rates and pathogen inoculum rates……………………………………………………….. 45 2.2.2 The experimental design, treatments, and data collection ………….. 50 2.3 Results ………………….……………………………………………... 56 2.4 Discussion and conclusions………………………..………………….. 70 References ………………………………………………………………… 75
v CHAPTER 3. THE NEMATODE APHELENCHUS AVENAE AND MYCOPHAGOUS COLLEMBOLAN AND MITES AS CONTROL METHODS FOR BELL PEPPER SEEDLING DAMPING-OFF CAUSED BY PHYTOPHTHORA CAPSICI...... 80 3.1 Introduction …………………………………………………………… 80 3.1.1 Bell pepper attacked by Phytophthora capsici and disease control…. 81 3.1.2 The specific objective ………………………………………………. 82 3.2 Materials and methods………………………………………………… 82 3.2.1 The preparation of nematode A. avenae, mycophagous collembolan and mites, and pathogen Phytophthora capsici, and determinations of germination rates and pathogen inoculum rates ………………….. 82 3.3 The experimental design, treatments, data collection and analysis …… 85 3.3.1 Experimental design ………………………………………………… 85 3.3.2 Experimental treatments……………………………………………... 86 3.3.3 Data collection …………………………………………..…………... 89 3.3.4 Statistical analysis …………………………………………………... 91 3.4 Results: ……………………………………………………………….. 91 3.4.1 Disease incidence and effects of treatments……………………….… 92 3.4.2 Effects of compost rate ……………………………………………… 94 3.4.3 Disease severity …………………………………………………….. 95 3.4.4 Pathogen activity ……………………………………………………. 96 3.4.5 Nematode populations ………………………………………………. 97 3.5 Discussion and conclusions …………………………………………… 99 References …………………………………………………………………. 103
vi LIST OF TABLES
CHAPTER 1. SOIL BIOLOGICAL INTERACTIONS AND BIOLOGICAL CONTROL OF FUNGAL PATHOGENS IN GREENHOUSE CROP AND FIELD TRANSPLANT PRODUCTION SYSTEMS Table 1.1 Microbial diversity in soil ………………………………………. 19
CHAPTER 2. BIOLOGICAL CONTROL OF DAMPING-OFF DISEASES OF GREENHOUSE VEGETABLE CROPS AND BEDDING PLANTS Table 2.1 ANOVA table for both tomato trials……….…………………… 57 Table 2.2 Relative activity of Pythium ultimum in growth chamber microcosms amended with the nematode Aphelenchus avenae, drenched with the fungicide mefenoxam, or left untreated……………….... 62 Table 2.3 ANOVA table for the bedding plants trials…………………….... 64 CHAPTER 3. THE NEMATODE APHELENCHUS AVENAE AND MYCOPHAGOUS COLLEMBOLAN AND MITES AS CONTROL METHODS FOR BELL PEPPER SEEDLING DAMPING-OFF CAUSED BY PHYTOPHTHORA CAPSICI Table 3.1 ANOVA table for pepper seedlings transplanted to infested field soil ……………………………………………. 92 Table 3.2 Average percentage of seedlings with symptoms per week following transplant to infested field soil …………… 93 Table 3.3 Relative activity of Phytophthora capsici for pepper seedlings transplanted to infested field soil in microcosms amended with the nematode Aphelenchus avenae, drenched with the fungicide mefenoxam, mycophagous collembolan, mites, or left untreated …………… 97
vii LIST OF FIGURES
CHAPTER 1. SOIL BIOLOGICAL INTERACTIONS AND BIOLOGICAL CONTROL OF FUNGAL PATHOGENS IN GREENHOUSE CROP AND FIELD TRANSPLANT PRODUCTION SYSTEMS Figure 1.1 Close-up picture of the mycelium of the basidiomycete soilborne pathogen Rhizoctonia solani showing the right angular branching and constrictions and cross-walls near junctions and the brown pigmentation that increases with age (EB Nelson, Cornell University) ………..………………... 5 Figure 1.2. Various biological interactions between pathogens and beneficial soilborne pathogens and their disease activities .…. 8 Figure 1.3. Soil mesofauna may play a major role in the suppression of soilborne pathogens and their disease activities .………...... 17 Figure 1.4. Diagram showing interactions between soil mesofauna and soil fungi ………………………………………………….. 24
CHAPTER 2. BIOLOGICAL CONTROL OF DAMPING-OFF DISEASES OF GREENHOUSE VEGETABLE CROPS AND BEDDING PLANTS Figure 2.1 Emergence of tomato seedlings in growth chamber microcosms: a) Trial 1 and b) Trial 2 …………………………. 59 Figure 2.2 Germination rates of tomato seeds as influenced by the rate of compost: a) Trial 1 and b) Trial 2 ………………..... 61 Figure 2.3 Average number of Aphelenchus avenae extracted from microcosms receiving the pathogen + nematode treatment, broken down by compost rate (%) in the second tomato trial ……………………………………………. 63 Figure 2.4 Disease incidence on vinca seedlings in greenhouse pots …….. 65 Figure 2.5 Disease incidence on impatiens seedlings in greenhouse pots ... 66 Figure 2.6 Disease incidence on vinca seedlings in greenhouse pots amended with 3 rates of compost; 4%, 8%, and 16% …………. 67 Figure 2.7 Disease incidence on impatiens seedlings in greenhouse pots amended with 3 rates of compost; 4%, 8%, and 16% …………. 68 Figure 2.8 Average number of Aphelenchus avenae extracted from pots receiving the pathogen + nematode treatment, broken down by compost rate (%) in the two vinca trials ………………….... 69 Figure 2.9 Average number of Aphelenchus avenae extracted from pots receiving the pathogen + nematode treatment, broken down by compost rate (%) in the two impatiens trials ………………. 69
viii CHAPTER 3. THE NEMATODE APHELENCHUS AVENAE AND MYCOPHAGOUS COLLEMBOLAN AND MITES AS CONTROL METHODS FOR BELL PEPPER SEEDLING DAMPING-OFF CAUSED BY PHYTOPHTHORA CAPSICI Figure 3.1 Damping-off disease incidence on pepper seedlings in infested field soil microcosms as affected by treatment………. 94 Figure 3.2 Damping-off disease incidence on pepper seedlings in infested field soil microcosms as affected by the rate of compost ………………………………………………... 95 Figure 3.3 Disease symptom severity as a function of time for treatment and compost rate combinations where pepper seedling damping-off symptoms caused by Phytophthora capsici appeared in infested field soil microcosms …………… 96 Figure 3.4 Average number of Aphelenchus avenae extracted from microcosms receiving the nematode treatment, broken down by compost rate (%): a) infested field soil and b) control field soil ……………………………………………. 98
ix Chapter 1. Soil biological interactions and biological control of fungal
pathogens in greenhouse crop and field transplant production systems
1.1 Greenhouse crop and field transplant production
1.1.1. The scope of greenhouse and transplant crop production
As the world population expands, and available land for crop growth decreases, there will be a growing need for increased productivity and year-round cultivation of crops (Omer,
2009). It will be necessary to extend the production time of crops past historical limits to meet the global demand for vegetable and other food crops, as well as ornamental horticultural crops. Greenhouses are microenvironments whose climates can be manipulated for plant growth and fruiting when atmospheric conditions in the field are not suitable.
Though initial investment of capital is high, greenhouse production allows both nutrient applications and watering regimes to be controlled by the grower and tailored to the needs of the specific crop being produced (Resh, 1987). Since the system is artificial and nutrient applications are specifically engineered by the grower, most nutrients are readily available for uptake by the plants, reducing competition and allowing for an increase in planting density (Paulitz, 1997). By controlling the climate and conditions, greenhouse crop production is advanced, can be sustained for an extended duration, and results in increased overall yield (Randal and Goyal, 1998). As a result of their increased crop production benefit, greenhouse use has increased greatly over the past decade to now cover hundreds of thousands of hectares of land worldwide (Yadav and Chauadhauri, 1997).
1 Greenhouse crop production is of significant agro-economic importance, especially in the southern United States. Major greenhouse crops include ornamental plants and vegetables grown both to maturity and to the seedling stage for transplant into the field. In North
Carolina, floriculture and nursery production ranks third in gross income behind only broilers and hogs, with a net income of over $870 million in 2005 (NCDA&CS, 2006). The floriculture industry includes production of bedding plants, propagative materials, foliage plants, cut flowers, potted flowering plants, and cut cultivated greens. Of these, the bedding plant segment is dominant, accounting for 55% of the industry (USDA-NASS, 2007).
Greenhouse vegetable production is a rapidly increasing industry, with about 800 acres of production in the United States (US Census of Horticulture Specialties, 2001). Over 80% of those 800 acres is dedicated to the growth of tomatoes, which are one of the most important cash crops in the U.S. In 2005, 37% of all fresh tomatoes sold in U.S. retail stores were greenhouse-produced, compared to negligible amounts in the previous decade (Cook and
Calvin, 2005). Many other vegetables and fruits are grown in greenhouses for ultimate transplantation for large-scale field production.
1.1.2. Soilborne fungi in greenhouse systems
The greenhouse crop and transplant production industry is significantly threatened by plant pathogens that attack the root system (Louws et al., 2005). Fungi and oomycetes are the most important soilborne pathogens, causing tissue death in the host plant (Agrios, 2005). In greenhouse production, seedling initiation and growth is subject to root rot problems, and in re-circulating systems, these pathogens can be spread easily from one plant to another and even transported to production houses. Also, the host plants are genetically identical and
2 therefore can be uniformly susceptible to infection by the pathogen. When these greenhouse production characteristics are combined with the dense planting of hosts, a small amount of contamination can lead to significant plant loss (Paulitz, 1997). In field production, seedlings transplanted to the field are most susceptible to many damping-off soilborne pathogens immediately following transplantation. The nutrients present in the rhizosphere allow many pathogens the opportunity to initiate a parasitic relationship with the host plant (Rovira and
Davey, 1974; Raajimakers et al, 2009).
1.2. Soilborne fungal pathogens of plants and their biological control
1.2.1. Soilborne fungal pathogens
Soilborne pathogens of greenhouse crops, including species of the fungal genera
Rhizoctonia, and the Oomycetes Pythium and Phytophthora can survive saprophytically or as dormant propagules in the absence of susceptible host plants (Agrios, 2005). Pathogens in these genera limit production and reduce economic profits in many plant industries, including agronomic crops, vegetable and fruit crops, turf, ornamental, and greenhouse crops. Both
Pythium and Rhizoctonia species cause root rot, and pre- and post-emergence damping-off in multiple species of plants (Haas and Keel, 2003). Phytophthora spp. also affect a wide range of crops, and specific pathogens, such as P. capsici, cause root, crown, and fruit rot in many highly valued crops (Hausbeck and Lamour, 2004; Louws et al., 2002).
Most Pythium spp. attack young plant tissue such as seedlings but some also can cause disease on stems and foliage in some hosts, including grasses, peanuts, and tomato transplants (Hendrix and Campbell, 1973). Damping-off of seedlings caused by Pythium spp. is most noticeable in nursery beds, field rows, and greenhouse flats because the disease
3 spreads and kills large areas of seedlings quickly. Pythium spp. can attack a wide range of hosts and can survive through saprophytic growth and resistant survival structures.
Saprophytically, P. ultimum is able to germinate and colonize new soil substrates rapidly due partly to its ability to grow under environmental conditions unfavorable to other organisms, including high soil moisture levels (Griffin, 1963). Resistant survival structures are much more important than saprophytic persistence for Pythium spp. in the soil. For short and intermediate time periods, survival is in the form of zoospores and sporangia, while long- term survival depends primarily on oospores (Thompson et al., 1971). Survival of P. ultimum sporangia has been shown to extend up to 11 months in soil and can germinate 1.5 hours after nutrient addition, reactivating their pathogenic capacity (Stanghelleni and Hancock, 1971).
Germination of the survival structure sporangia through germ tubes or zoospores allows P. ultimum to attack host plants including tomatoes. Once in a mycelia state, three possible pathways exist for P. ultimum: i) lysis, ii) infection of a susceptible host, or iii) formation of new sporangia as survival structures. Lysis and formation of new sporangia for survival occurs when there is no suitable food source for mycelia growth and it has been shown that the pathogen P. ultimum grows more abundantly in the rhizosphere of susceptible host plants than in that of nonsusceptible plants (Alicbusan et al., 1965).
Rhizoctonia diseases occur worldwide on almost all vegetables and flowers, multiple field crops, turfgrasses, ornamentals, shrubs, and trees. Damping-off, root rot, stem rot and stem canker are also widespread symptoms caused by Rhizoctonia. Occurring mostly in cold, wet soils, damping-off attacks young seedlings both before and after emergence from the soil
(Agrios, 2005).
4 The genus Rhizoctonia is composed of a widely diverse group of organisms.
Rhizoctonia exists mostly as sterile mycelium with only rare production of spores in nature.
The mycelium begins growth colorless, and gains yellow or brown pigment with age. As the often solitary means of identification, a unique characteristic is how the hyphae branch in a distinct right angle to the main hypha, with slight constrictions and cross walls near their junctions (Agrios, 2005)(Figure 1.1). When the rare production of spores occurs, it is in the form of basidiospores.
Figure 1.1. Close-up picture of the mycelium of the basidiomycete soilborne pathogen Rhizoctonia solani showing the right angular branching and constrictions and cross-walls near junctions and the brown pigmentation that increases with age (EB Nelson, Cornell University).
Another unique aspect of R. solani is the anastamosis group (AG) concept.
Anastomosis by definition means fusion of touching hyphae, and since this can occur only
5 between isolates within the same AG group, it is a means of distinguishing one R. solani strain from another. It has been determined that R. solani is not a single species like most soilborne plant pathogens, but rather is a collection of non-interbreeding populations that are recognized using this AG concept (Anderson, 1982). The AG concept was initially proposed by the German scientist H. Schultz in 1937 and developed in the US by Parmeter and colleagues beginning in 1969. The existence of multiple anastomosis groups within R. solani indicates genetic isolation of the populations in each group (Agrios, 2005).
Host specificity of R. solani is also linked to these anastomosis groups. There are more than 10 identified anastomosis groups within R. solani, with AGs 1-4 the most common. AG-1 isolates cause seed and hypocotyls rot and sheath and web blights of many different plant species. The isolates of AG-2 cause root crop cankers, wire stem on crucifers, and brown patch on turfgrass. Potatoes are the most affected by isolates of AG-3, though the symptoms range from stem canker to black sclerotia production on tubers. Finally, isolates of
AG-4 infect a wide range of host plants, causing seed and hypocotyls rot and stem lesions
(Agrios, 2005).
Along with many other Phytophthora spp., P. capsici is capable of causing multiple cycles of disease within a single growing season (Ristaino, 1991). The optimum growth temperatures for P. capsici are 25-28 ºC and under this condition large numbers of sporangia are produced on infected tissue surfaces. Once they come into contact with host plant tissue, the zoospores encyst and produce germ tubes by which they can directly penetrate the plant’s cuticle as quickly as an hour (Hausbeck and Lamour, 2004). The penetration through the host
6 plant cells may be direct or through natural openings such as stomata (Yoshikawa et al.,
1977).
It has been reported that host plants are more susceptible to P. capsici as seedlings than as mature plants (Kim et al., 1989). Damping-off of bell pepper seedlings due to P. capsici is a major problem in vegetable production fields and greenhouses.
1.2.2. Biocontrol of soilborne fungal pathogens
The original definition of biological control (biocontrol) in terms of plant pathogens was coined by Cook and Baker (1983); “the reduction of the amount of inoculum or disease- producing activity of a pathogen accomplished by or through one or more organisms other than man”. The goal of biocontrol is to augment a natural phenomenon or process to suppress pathogens and their activities through the manipulation of the host, pathogen, environment, or macro- or microflora or fauna of the system of interest. In basic terms, there are two main types of biocontrol; general suppression and specific suppression (Hoitink et al., 1997).
General suppression refers to pathogen and/or disease suppression resulting from the collective activities of the soil microbial community. One common practice is to utilize soil amendments that enhance the growth of beneficial microorganisms that in turn help suppress detrimental soil pathogens that cause a wide range of diseases in the host plants. Specific suppression is the targeted application of a known biocontrol agent for suppression of a specific pathogen or disease.
7 The major biocontrol mechanisms identified include competition for C and nutrients, antibiotic responses of co-existing microorganisms, hypovirulence, and parasites on specific pathogens or induced resistance in plants by beneficial microorganisms (Duso et al., 2005;
Kerr, 1980; Kloepper et al., 2004) (Figure 1.2).
Figure 1.2. Various biological interactions between pathogens and beneficial microbes contribute to the suppression of soilborne pathogens and their disease activities.
Rouse and Baker (1978) found that Rhizoctonia root rot of bean was suppressed when the soil was amended with cellulose. They hypothesized that the cellulose amendment in the soil was utilized by the beneficial microorganisms, increasing their population size and out- competing the pathogenic Rhizoctonia solani for N. The pathogen requires N to germinate
8 and as a result of the competition by the beneficial soil microorganisms, the R. solani was unable to cause disease (Rouse and Baker, 1978).
Also, many microorganisms produce antibiotic compounds that inhibit other microbes to gain a competitive edge for resources. Some biocontrol agents utilize antibiosis via production of compounds inhibitory to plant pathogens. The most well-known specific biocontrol agent, Agrocin 84, is a compound produced by the non-pathogenic Agrobacterium radiobacter K84, which blocks the synthesis of DNA by the pathogen Agrobacterium tumefaciens (Kerr,
1980). The causal agent of crown gall in a wide range of woody host plants from apples to roses, Agrobacterium tumefaciens is a soilborne bacterial pathogen that is now consistently controlled with the biocontrol agent Agrocin 84.
Hypovirulence is a biocontrol mechanism where genetic exchange between a hypovirulent form of a pathogen in natural population with a virulent form reduces the virulence of the pathogen. Some hypovirulent strains of the Hypoviridae family are capable of reducing the virulence of the ascomycete fungal chestnut blight pathogen Cryphonectria parasitica via horizontal gene transmission (Van Alfen et al., 1975).
In parasitism, one organism can directly parasitize another organism or species. In one specific form of parasitism that has been utilized in biocontrol, the biocontrol agent is able to directly parasitize the pathogen’s hyphae or other propagules resulting in suppression of the pathogen and disease. Species of the genus Trichoderma are among the most well-studied biocontrol agents, dating back to T. lignorum mycoparasitizing R. solani, the causal agent of citrus seedling disease in 1932 (Weindling). Weindling (1932) described how the hyphae of
9 the biocontrol agent T. lignorum coiled around the R. solani hyphae, directly penetrated it, and dissolved the cytoplasm within its cells.
Finally, induced resistance is a relatively new addition to the list of biocontrol mechanisms of plant pathogens and their diseases. Though induced resistance can utilize multiple pathways including jasmonic acid and salicylic acid, the concept falls under the single concept of “active resistance dependent on the host plant’s physical or chemical barriers, activated by biotic or abiotic agents (inducing agent)” (Kloepper et al., 1992). Many chemicals or organisms can function as inducing agents including microorganisms, and phosphate and organic nutrients, triggering signaling cascades that lead to resistance to pathogens in the host plant (Lugtenberg and Kamilova, 2009).
Many strategies have been tested for the control of soilborne plant pathogens based on the introduction of a single or multiple biological agents. However, this approach to soilborne pathogen control has not been widely adopted for several reasons (Hoitink and
Boehm, 1999). Some introduced agents have only been effective against a single pathogen and/or disease but were unable to control multiple diseases of important crops. Others have only provided partial control of the disease, or have not been able to survive long enough to provide any control at all (Weller, 1988). To address these issues, factors affecting a biocontrol agent’s ability to establish on plant roots have been studied.
Rhizosphere competency of a biocontrol agent is important because the rhizosphere is the place where disease suppression is most needed and where food is readily available for the agent (Harman, 1992). Plant roots exert influence over the rhizosphere via root exudation, production of mucilages and releasing sloughed-off root cells and by providing an ecological
10 niche for soil microorganism growth (Bais et al., 2006). The same rhizosphere that is utilized by soilborne pathogens as their primary habitat is home to microbial groups and other agents including bacteria, nematodes, fungi, microarthropods, and protozoa. Many of these rhizosphere-inhabiting microorganisms have a neutral effect on the host plant, but some have a beneficial effect, including nitrogen-fixing bacteria, plant growth-promoting rhizobacteria, and endo- and ectomycorrhizal fungi (Raajimakers et al., 2009). Additionally, some of these rhizosphere-inhabiting microorganisms can have an antagonistic effect on the soilborne pathogens themselves in various ways (Buee et al., 2009). Biocontrol microorganisms can adversely affect soilborne pathogens’ population densities, spatial and temporal dynamics, and metabolic activities through three main mechanisms; competition, antagonism, and hyperparasitism (Raajimakers et al., 2009). There are over 80 products commercially available for biocontrol of plant pathogens worldwide currently, with most consisting of a formulation of either the fungi Gliocladium-Trichoderma or the bacterial species
Pseudomonas and Bacillus (McSpadden Gardener, 2010). Of these products, some are registered not as biocontrol agents, but as plant growth-promoters, plant strengtheners, or even as soil conditioners as a way of circumventing the stringent efficacy and toxicology testing required for plant protectant products (Paultiz and Belanger, 2001). For example, some fluorescent Pseudomonas PGPR strains are capable of producing iron-sequestering siderophores, rendering the soft-rot plant pathogen Erwinia carotovora incapable of growth
(Kloepper et al., 1980). An area that has to-date been largely under-investigated is the role of soil fauna in the interactions between plant pathogens and host plants in the rhizosphere
(Whipps, 2001).
11 The control of soilborne pathogens has traditionally relied on the use of cultural practices such as crop rotation and no-tillage, and the application of fungicides and fumigants
(Reeleeder 2003; Martin 2003). Broad-spectrum fumigants like methyl bromide have been effective in controlling soilborne pathogens in high-value crops including tomatoes and bell peppers in the past but methyl bromide has proven to be dangerous to both humans and the environment and as such is being phased out of use completely (Horrigan et al., 2002; Martin
2003). Fungicide movement in soil is often limited by the soil matrix or pore availability, and soil leaching and chemical breakdown by microbial communities also alter fungicide efficacy
(Paulitz, 1997). Fungicides are commonly applied multiple times for plant disease control in conventional agriculture systems, but these synthetic pesticides have also been shown to negatively affect the health of the ecosystem (Echobichon, 2001; Agrios, 2005). Most fungicides also require a restricted-entry period post-application before workers can return to the treated crop, and a similar harvest interval is also commonly required between last application and harvest of the crop. Since many greenhouse crops are continuously harvested, fungicide application as part of a disease management strategy often is not possible (Paulitz and Belanger, 2001). Additionally, heavy utilization of fungicides greatly increases production costs for growers and producers and can result in the development of resistant pathogen populations. Overall public awareness of the environmental impact of chemical pesticides in agriculture has been shifting the focus away from high-energy input agroecosystems towards more ecologically sustainable systems. In these systems, synthetic chemicals are at the least minimized, and biologically-based processes are used for both nutrient supply and pathogen control (Tilman et al., 2002). Equally important, there are few
12 fungicides or other chemical products currently registered for use in greenhouse crop production systems against root diseases on food or horticultural crops (Paulitz and Belanger,
2001).
Since Pythium spp. cause a variety of diseases in a wide range of important crops, efforts to control the pathogens with biological control have been studied previously. Pythium oligandrum Drechsler is a vigorous necrotrophic mycoparasite active against other Pythium species, including the soilborne pathogen P. ultimum (Drechsler, 1943). As a biocontrol agent applied as a cress seed coating, P. oligandrum Drechsler provided protection against damping-off caused by P. ultimum (Al-Hamdani et al., 2003). Bacteria have also been tested for biological control ability against Pythium spp., with specific focus on fluorescent
Pseudomonads due to their rapid growth rate and ability to widely colonize the rhizosphere
(Fukui et al., 1994). The mechanism of biocontrol for these Pseudomonads is mainly competition for the limited rhizosphere carbon, and the production of secondary metabolites such as antibiotics, siderophores, and cyanide that are antagonistic to the Pythium species
(O’Sullivan and O’Gara, 1992). Compost amendments have also been studied for their effectiveness in suppression of Pythium spp. that cause seedling damping off. Scheuerell et al., (2004) tested a wide range of composts for their ability to suppress damping off of seedlings caused by both P. ultimum and R. solani and found that significant disease suppression was achieved by multiple compost blends. There has been little to no work done on the use of mycophagous soil mesofauna as biocontrol agents for the control of damping- off caused by P. ultimum.
13 Rhizoctonia solani is well-studied as a plant pathogen, due in part to widespread distribution in cultivated and non-cultivated soils and its ability to infect a wide range of host plants, as well as the lack of an effective disease management strategy (Faltin et al., 2004).
Many researchers have concentrated on biological control as an alternative disease management strategy for this pathogen. Multiple studies have also been performed on suppressive soils and soil amendments as a means of controlling disease caused by R. solani.
Suppression of diseases caused by R. solani has been reported in both field and potting soils. For example, Roget (1995) reported a natural decline in cereal disease caused by R. solani. Though the disease incidence rapidly increased the first 3 years, during the next
8 years, disease incidence decreased down to an ultimately undetectable level. In a follow-up study, the suppression of wheat root rot caused by R. solani AG-8 was determined to be biological in nature (Wiseman et al., 1995). Damping-off caused by R. solani is a major disease problem in commercial greenhouse production of bedding plants such as vinca and impatiens (Cline et al., 1988).
Suppression of pre-emergence damping-off of impatiens seedlings caused by R. solani has been reported in potting soils amended with animal manure composts. Diab et al.
(2003) conducted an experiment on the effect of different rates of composted swine waste- amended peat-based potting soil on the suppression of disease on impatiens bedding plants.
Their results showed that pre-emergence impatiens seedling damping off was lower in composted swine waste-amended potting soil than non-amended potting soil. The disease suppression was attributed to enhanced microbial activity as a result of the compost
14 amendment. My thesis research utilized dairy cow compost-amended potting soil in hopes of stimulating mycophagous mesofauna activity for disease suppression.
Most of the biological control agents for R. solani belong to the genera Trichoderma,
Gliocladium, Cladorrhinum, and binucleate Rhizoctonia; relatively few bacteria have also been identified as effective. Non-pathogenic binucleate Rhizoctonia has been shown to be effective in suppressing diseases caused by pathogenic Rhizoctonia species in a variety of ornamental plants and crops, as well as suppressing diseases caused by Pythium species
(Honeycutt and Benson, 2001). Non-pathogenic binucleate Rhizoctonia was also used in our research, albeit as a food source for the mycophagous nematode Aphelenchus avenae.
One study conducted by Ahmed and colleagues in 2003 showed that it was the combination of amending the soil with chitin and the biological control agent T. harzianum that effectively suppressed R. solani following a single seed and root drench. Their results suggested that the addition of chitin stimulated the inhibitory effects of the fungi to levels of suppression not achieved by the fungi alone (Ahmed et al., 2003). The addition of a soil amendment for use in support of the main antagonistic biocontrol agent against soilborne pathogens is similar to the design of my thesis research experiments. Instead of using a non- pathogenic fungal strain for disease suppression, mycophagous mesofauna were used as the main biological control agents.
Many species of the bacterial genus Pseudomonas have been studied for their applicability as biocontrol agents for the control of plant pathogens. One reason for their popularity is the production and excretion of chitinases and !-glucanases. These enzymes
15 digest the cell wall of fungal propagules for use as carbon and food sources (Gooday, 1990).
Thus, the inoculum density of the pathogen in the soil is reduced, suppressing disease of the host plant. In 2007, Arora et al. reported findings showing Pseudomonas GRC3 strongly inhibited the growth of the soilborne fungal pathogens Rhizoctonia solani and P. capsici and determined the mechanism of inhibition to be antibiosis.
Fungi have also been studied for their biocontrol activity against P. capsici. Another much-studied biocontrol agent, the fungus Trichoderma harzianum, significantly reduced root rot caused by P. capsici when both were present in the same substrate (Ahmed et al.,
1999). This reduction in disease was attributed to the reduction of pathogen population growth of Phytophthora capsici by T. harzianum. The P. capsici population growth reduction was demonstrated both in vitro and in vivo. The antagonistic effect on P. capsici was attributed to T. harzianum out-competing the pathogen for nutrients (Ahmed et al., 1999).
Soil amendments for the suppression of disease caused by P. capsici have also been studied. In 2008, Liu et al. reported that disease onset and suppression of bell pepper caused by P. capsici was related to the type of amendment used as well as changes in soil physical properties. The amendments also affected the soil respiration and microbial biomass C and
N. However, little to no research has been performed on the control of Phytophthora capsici causing bell pepper seedling damping off using mycophagous soil mesofauna.
16 1.3. Soil food web interactions and biocontrol
1.3.1. Soil mesofauna and soil food web interactions
Food web interactions among soil biota can significantly affect plant growth through regulating nutrient availability for plants and impacting the incidence of soilborne plant pests and diseases (Brussaard et al., 2007)(Figure 1.3).
Figure 1.3. Soil mesofauna may play a major role in the suppression of soilborne pathogens and their disease activities.
The functionality and dynamical behavior of soil is a consequence of the interactions among the chemical, physical, and biological processes as mediated by the soil structure
(Young and Crawford, 2004). Trophic dynamics govern the movement of energy, C, and
17 nutrients among organisms in an ecosystem. A trophic level is comprised of organisms that obtain their energy with the same number of transfers from plants or detritus and occupy the same position in the food web. Therefore, a food web is a complex comprised of linked food chains of organisms that linearly transfer energy and nutrients. There are two main food web interactions: bottom-up control where the availability of food at the base of a food chain limits the production of upper trophic levels, and top-down control where predators regulate the abundance and dynamics of their lower trophic level prey (Chapin et al., 2002).
Soil trophic interactions can exert significant effects on plant growth through altering nutrient availability for plants and/or microbial associations with plants. The main biotic factors that influence plant interactions and coexistence are niche differentiation, competition, predation/herbivory, mutualism, and parasitism (Reynolds et al., 2003). Soil communities belowground and host plants aboveground are interdependent, and as such, soil mesofauna and their food web interactions can have a great impact on the growth and development of plants (Bever, 2003). Soil microorganisms, including mesofauna play a variety of roles in ecosystem functioning; decomposition of organic matter, nutrient cycling, bioturbation, and suppression of soilborne diseases and pests (Brussaard et al., 1997). Soil bacteria and fungi are responsible for 90% of the mineralization of carbon, while soil mesofauna shred residue and disperse microbial propagules together forming the decomposers of the soil (Chapin et al., 2002). Soil organisms play key roles in the ecosystem functioning through facilitating nutrient cycling and maintaining water quality. Larger soil animals and plant roots are responsible for creating channels, pores, and aggregates in the soil to allow the transport of gases and water as well as providing habitats for smaller soil
18 microorganisms. These bioturbators are essential for maintaining soil structure (Brussaard et al., 1997). The diversity of soil microorganisms is thought to be extremely high, but is still largely unidentified. The number of bacterial species per gram of soil is in the range of hundreds of thousands with total bacterial species estimated in the millions (Gans et al.,
2005). Similarly, species diversity of soil fungi is thought to be only slightly lower in number
(Bridge and Spooner, 2001). In natural ecosystems such as forest soils, hundreds of species and thousands of individuals are concentrated in the litter layer and uppermost mineral-soil layer (Hattenschwiler et al., 2005). Both the diversity within and between trophic groups are high in the soil environment. Among soil invertebrates, fungivores (Collembola, Oribatida,
Nematodes) are the most highly abundant and usually dominate soil communities in terms of species numbers (Coleman and Crossley, 1996) (Table 1.1).
Table 1.1. Microbial diversity in soil.
Organism Approximate number of documented species globally Bacteria 10000 Fungi 72000 Nematodes 25000 Collembola 7500 Mites 45000
Adapted from DeDeyn and Van der Putten, 2005.
As an ecosystem, soil is considered healthy when it demonstrates the following characteristics: (1) integrity of nutrient cycles and energy flows, (2) biological diversity, (3) interconnectedness between functional units, (4) stability and resilience when faced with disturbance or stress, and (5) limited plant disease outbreaks (Rapport, 1995). To maintain
19 soil health it is necessary to promote high primary productivity and high microbial biomass, activity and diversity. In general, natural soils are considered healthier than cultivated soils as cultivated soils usually have lower microbial diversities and more severe disease problems than they had as natural habitats (Ko, 1982). High microbial biomass, activity and diversity in soil have been associated with the suppression of soilborne plant diseases in organic soils
(Mader et al., 2002). In fact, soils with a high biological diversity and activity, such as natural or organically-amended agricultural soils are frequently more suppressive to soilborne diseases than conventionally-managed agricultural soils (van Bruggen, 1995).
Nematodes are the most abundant and microarthropods are the most diverse animals in the soil, capable of reaching populations in excess of 106 and 105 individuals per m2 respectively (Brussaard et al., 1997). Mesofauna, including nematodes and collembola, feed on both bacteria and fungi in the soil (Coleman and Crossley, 1996). This top-down control via faunal predation is a major mechanism through which essential plant nutrients are released from microbial biomass and made available for plant uptake (Coleman et al., 1983;
Cole et al., 2004). Grazing of pathogenic soilborne fungi by generalist predators also provides a potential disease suppression mechanism (Beute and Benson, 1979; Bollen et al.,
1991; Friberg et al., 2005). Fungal-feeding collembola graze directly on fungal hyphae and consume fungal spores and other propagules, while fungal-feeding nematodes penetrate fungal cells with their mouth stylet and ingest the contents (Friberg et al., 2005). Suppression of fungal pathogens and their activities has mostly been investigated using organisms of the same trophic level (Martin, 2003; van Bruggen et al., 2006). Only a few studies have
20 examined the feasibility of using soil mesofauna for disease suppression and the practicality of such studies has not been investigated (Friberg et al., 2005).
In addition to water and temperature, nutrients are the factor that most constrains terrestrial plant growth. The productivity of all ecosystems responds to the addition of one or more nutrients, indicating widespread nutrient limitation (Lambers et al., 2009). In conventional agricultural systems, microbial biomass is low because of the low availability of C and the high rate of turnover and physical and chemical disturbance. This low C availability and thus low fungal biomass cannot support a large enough population of fungal- feeding nematodes and collembola to result in suppression of pathogen growth and disease development (Bandyopadhyaya et al., 2002; Cassagne et al., 2003). One important approach to biological control of soilborne plant pathogens is the introduction of a food base with the biocontrol agent that supports the agent’s activity without also stimulating pathogen activity
(Hoitink and Boehm, 1999). For example, incorporation of composts into potting media increases soil microbial biomass and fungal dominance (Boehm and Hoitink, 1992).
Compost is a “product of biological decomposition and stabilization of organic substrates under conditions that allow thermophilic temperatures as a result of biologically-produced heat, to produce a final product that is stable, free of pathogens and plant seeds, and can be beneficially applied to land” (Haug, 1993). The organic substrates used for compost formation can include green wastes such as grass clippings, food waste, industrial waste, crop residue, animal manure, and sewage sludge. Through enhancing the response and function of organisms in the soil food web, organic matter amendments improve soil structure and physical conditions; cation exchange capacity, moisture retention, crop production including
21 overall health of host plants and yield, and soil biological activity including mesofauna population growth and sustainability (Ferris and Matute, 2003; Agnew and Leonard, 2003).
The composting process can be dividing into 3 phases with different operating temperatures and compounds and organisms incapacitated at each phase. The first phase occurs during the initial 24-48 hours with sugars and other easily biodegradable substances are destroyed as a result of temperatures in the range of 40-50 ºC. Next, the temperatures rise to 55-70 ºC. Most plant pathogens and weed seeds are killed in this stage, and other less biodegradable cellulose-based substances are destroyed (Bollen, 1993). In order to expose all parts of the compost to these necessarily high temperatures, compost piles must be turned frequently.
The final curing stage consists of lower temperatures, and decreased temperature outputs and decomposition rates as the concentration of readily biodegradable components in the wastes decreases (Hoitink et al., 1997).
Organic matter amendments improve soil structure and physical conditions, cation exchange capacity, moisture retention, crop production including overall health of host plants and yield, and soil biological activity including mesofauna population growth and sustainability (Ferris and Matute, 2003; Agnew and Leonard, 2003). In addition to creating a more beneficial environment for both the plant and the soil organisms, composts may be suppressive to disease-causing soilborne pathogens including species of the genera Pythium,
Phytophthora, and Rhizoctonia through enhancing general microbial activities (Hoitink and
Fahy, 1986; Hoitink and Boehm, 1999). This type of biological control is based on activities of the agents within the context of the soil microbial communities and their responses to soil and plant-related energy reserves (Hoitink et al., 1997). Additions of organic materials,
22 including animal manure, in general enhance microbial biomass, activity and diversity, and food web complexity in soil (Mader et al., 2002). Microorganisms stimulated by these organic amendments also contribute to the suppressive activity of the amended soils through all four principal mechanisms of biological control (van Bruggen, 1995): (1) competition, (2) antibiosis, (3) parasitism/predation, and (4) induced systemic resistance (Lockwood, 1988).
These organic amendments also enhance the suppression of many soilborne plant pathogens.
For example, propagule densities of Phytophthora and Pythium were lower and that of antagonist Trichoderma was higher in soils amended with various organic materials than soils with synthetic fertilizer (Bulluck et al., 2002).
1.3.2. Soil mesofauna as biocontrol agents
Interactions between soil invertebrates and plants, mediated by soil microorganisms are numerous and important (Scheu, 2001). Soil mesofauna indirectly influence host plant – pathogen relationships by altering the physical soil environment by fragmenting organic matter, burrowing through the soil, soil mixing, and deposition of their feces. More directly, soil meosfauna influence pathogenic fungi and other soil microbes by feeding on them and dispersing them (Curl, 1988) (Figure 1.4).
23
Figure 1.4. Diagram showing interactions between soil mesofauna and soil fungi. Highlighted in green are the 3 categories of soil mesofauna focused on in this paper. The thick arrows indicate flow of energy as a result of mycophagous soil animals grazing on fungi. The thin arrows indicate positive influences caused by these interactions. Adapted from Krivtsov et al., 2004.
Mesofauna play different roles in soil communities based on their food preference and feeding mode and are classified as fungivorous, bacterivorous, omnivorous, predatory, or plant parasitic (Nicholas, 1984). Fungivorous soil mesofauna feeding on saprophytic fungi can increase the amount of nutrients in the soil that may otherwise be rendered unavailable for plant uptake in the fungal biomass (Hanlon, 1981). It has been shown that increased nutrient availability in the presence of fungal grazers is concurrent with plant growth enhancement (Setala and Huhta, 1991).
The two main biological control approaches using soil mesofauna are 1) incorporating soil organic materials that promote growth of the pre-existing populations of
24 soil mesofauna antagonistic to plant pathogens and 2) adding cultures of specific highly active soil mesofauna that are antagonistic to plant pathogens in soil (Barnes et al., 1981).
Fungivorous, or mycophagous, nematodes can act as biocontrol agents of the fungal pathogens Fusarium, Pythium, and Rhizoctonia (Okada, 2006). The most common fungivorous nematodes in soil include species of the genera Aphelenchus, Aphelenchoides,
Ditylenchus and Tylenchus (Freckman and Caswell, 1985). Fungivorous nematodes are usually polyphagous, meaning they feed on a wide range of soil fungi, include saprophytic, pathogenic, and mycorrhizal fungi (Ruess and Dighton, 1996). Feeding on different fungi affects the soil community and host plants differently. Feeding on pathogenic fungi can lead to disease suppression, but feeding on mycorrhizal fungi can reduce their beneficial effects for the host plant. In a natural soil environment, more than one type of fungus can be present as a potential food source for fungivorous nematodes. Several studies have found that fungivorous nematodes preferentially feed on pathogenic soil fungi when presented with saprophytic, and pathogenic fungi (Ruess and Dighton, 1996). The fungivorous nematode
Aphelenchus avenae is found worldwide and can feed on over 90 species of fungi from 50+ different genera (Townshend, 1964; Barnes et al., 1981). Aphelenchus avenae has also been shown to prefer plant pathogenic fungi to saprophytic fungi as a food source in soil (Mankau and Mankau, 1963). This feeding preference for plant pathogenic fungi, its ubiquitous presence in soil, and its non-plant-parasitic status make Aphelenchus avenae an excellent candidate for biological control of soilborne fungal pathogens and their diseases (Steiner,
1936).
25 Collembola, or springtails, are soil arthropods that are also widespread and abundant in soils and leaf litter. Like Aphelenchus avenae, they are capable of feeding on fungi and are not plant-parasitic (Christiansen and Bellinger, 1981). Fungivorous collembola have been observed to graze on hyphae of arbuscular mychorrizal fungi as well as saprophytic fungi and root pathogenic fungi (Klironomos et al., 1992). Collembola also graze preferentially on fungi, with root pathogens being most preferred, then saprophytic fungi, and arbuscular mycorrhizal fungi being the least preferred (Lartey et al., 1994; Klironomos and Kendrick,
1996). In growth chamber experiments, a reduction in disease severity caused by Rhizoctonia solani, Pythium ultimum, and Fusarium oxysporum f. sp. cucumerinum has been shown as a result of the feeding activity of collembolan (Curl et al., 1985; Bollen et al., 1991; Lartey et al., 1994; Lootsma and Scholte, 1997a; 1997b; 1998).
Mites of the taxonomic group Oribatid (Acari:Orbatida) are also classified as feeders of fungi (Krantz, 1978). Research has demonstrated that population levels of mites are directly correlated with disease suppression of several important host plants. Mendel and
Gerson (1982) showed that sooty mold increased on citrus where the tydeid mite Lorryia formosa Cooreman had been removed with an acaricide. Abundance of the tyedid mite
Tydeus caudatus Dugés was positively correlated with the severity of grape downy mildew in
Italian vineyards (Duso et al., 2005). Similar to nematodes and collembolan, mites are widespread and have a broad habitat range (English-Loeb et al., 2007).
26 1.4 Integrated biological control of fungal pathogens and diseases in greenhouse and transplant crops: Opportunities and challenges
1.4.1 Fungal-feeding animals and soil amendments
The extensive hyphae of fungi, including that of plant pathogenic species, constitute a major component of the microbial biomass present in the soil. In arable and forest ecosystems, fungal hyphae can extend up to 400m and 2000m per gram of soil respectively
(Christensen, 1989). In a soil ecosystem, fungi are involved in many complex interactions with plants, detritus, animals, bacteria and the abiotic environment (Carroll and Wicklow,
1992). All soil animal taxa contain fungal-feeders, including nematodes and collembolans and play a significant role in the release of nutrients to both host plants and beneficial mycorrhizae (Klironomos and Hart, 2001). In preferential feeding experiments with multiple food choices, fungivores have shown consistently similar food preferences over a range of animal taxa with plant pathogens being the most preferred fungal food source and mycorrhizae being the least preferred (Bonkowski et al., 2000). Soil enrichment with a specific ingredient, such as compost, is a means of supporting a community of beneficial soil microorganisms for the benefit of the host plant and suppression of pathogens. The mechanism of disease suppression by organic amendment is primarily due to the provision of suitable niches and enhancement of edaphic microorganisms or parasitism and/or antibiosis of antagonists for potential biocontrol (Fukui, 2003). This type of soilborne disease control is important because it utilizes natural resources without chemicals. In addition to disease control, the use of organic amendments provides nutrients to crops and organic matter to the soil, and enhances the complexity of soil community and general soil biological activities.
27 In the United States there are over 1.5 million farms, with over 50% of those farms classified as animal production. These animal production farms raise six categories of livestock; cattle, hog and pig, poultry, sheep and goat, animal aquaculture, and other animal production (Profile of the Agricultural Livestock Production Industry, 2000). A by-product of the livestock industry is the issue of waste disposal. One disposal method that has been developed and is increasing in popularity is using animal waste to produce compost. In North
Carolina, hog, poultry and cattle are the top three livestock produced on animal production farms. The Smith family of Daddy Pete Farms in Stony Point, NC produces organic products on their 250 acres of land, including several compost mixes. Daddy Pete’s Plant Pleaser
Dairy Cow Manure is an organic compost that is a 100% natural fertilizer made in North
Carolina and available around the country (Daddy Pete Farms, Stony Point, NC).
1.4.2. Biological control in greenhouse and transplant crops
The absence of fungicides registered for greenhouse use, and the inability to employ conventional agricultural cultural practices make controlling soilborne diseases in greenhouse and transplant crop production difficult. When coupled with the growing desire for non-chemical based integrated pest management strategies, alternative disease control systems for greenhouse use are in urgent need (Paulitz and Belanger, 2001). While high humidity, warm temperature, high soil moisture content, and other greenhouse conditions are conducive to disease development, they are also ideal for the growth of antagonistic microorganisms suitable for use as biological control agents (Loper and Stockwell, 2000;
Kloepper et al. 2004). Like soilborne pathogens, biocontrol agents are sensitive to environmental conditions which can be optimized to favor each agent specifically (Paulitz
28 and Belanger, 2001). The logistics and economics of biocontrol applications are more suited to greenhouse use than field application in general. Since greenhouse crops have a higher economic value, and are produced in greater densities than field crops, the potentially higher cost of biocontrol agents as part of a disease control regime would not reduce gross income as much as in field crop application. Also, as a result of greater density of plants, the amount of biocontrol agent inoculum applied in greenhouse crop production would be greatly reduced compared to the amount applied at the same rate in field crop production (Paulitz and Belanger, 2001). Biocontrol in greenhouse systems has gained an advantageous niche as a by-product of the negative public perception of chemical applications in food crop production. Organic or pesticide-free vegetables and ornamentals are in growing demand with consumers year-round, giving more support to the development of biocontrol disease management systems. Finally, as greenhouse growing systems are inherently more technologically-based, growers may be more accepting of biological control management systems than more conventional agricultural systems with traditional management (Paulitz and Belanger, 2001).
1.4.3 Integrated biological control
An integrated pest management system that is environmentally friendly and suitable for use in greenhouse and transplant systems to control soilborne fungi is important and urgently needed. Utilization of dairy cow compost in conjunction with native soil mesofauna populations to graze directly on the soilborne pathogens may provide a novel approach for pathogen and disease control. When incorporated into potting soil, dairy cow compost not only enriches the plant growth substrate, but also may also provide organic matter to support
29 the microbes and the soil food web. Increasing the food source for the soil mesofauna will allow them to grow and reproduce to population levels capable of providing control of the soilborne fungal pathogens.
1.4.4. The overall goal and specific objectives of this research
The overall goal of my research was to evaluate the efficacy of fungal-feeding nematodes, collembola, and mites as an alternative to chemical controls for soilborne fungal pathogens and their diseases in greenhouse and transplant crops.
The specific objectives of this research were to:
A. Evaluate the efficacy of the nematode Aphelenchus avenae, and the unidentified
collembolans and mites in controlling the disease activities of the three fungal
pathogens Pythium ultimum, Rhizoctonia solani, and Phytophthora capsici in
greenhouse crops
B. Determine whether the enhancement of fungal-feeding nematodes, collembolan, and
mites using dairy cow compost in the rhizosphere of seedlings can effectively
suppress fungal pathogens and their disease activities
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41 Chapter 2. Biological control of damping-off diseases of greenhouse
vegetable crops and bedding plants
2.1 Introduction
Damping off of seedlings is commonly caused by Pythium spp., Phytophthora spp. and Rhizoctonia solani (Agrios, 2005). The disease can affect seeds, seedlings, and roots of plants before or after seedling emergence. Pre-emergence damping-off results in poor germination of seeds or poor emergence of seedlings. Post-emergence damping-off results in complete destruction of the seedlings in the seedbed or death immediately following emergence or transplant (Dhingra et al., 2004). Young seedlings can be attacked before emergence at any location on the plant, with the infection spreading rapidly onward causing invaded cells to collapse and the seedling to die (Agrios, 2005). The infected cells become waterlogged and discolored, and collapse easily. The infected part of the seedling stem close to the soil line becomes softer and thinner compared to the healthy tissue above it and thus the seedling falls over onto the soil. The fungus or fungal-like pathogen can then continue to invade the healthy seedling tissue, causing the fallen seedling to wither and die. Both pre- and post-emergence seedling damping-off can result in reduced yields (Begum et al., 2010).
Bedding plants and transplant vegetable crops treated with biocontrol agents during greenhouse production time may carry the biocontrol agent along on the root system with them to the field. Thus, if the biocontrol agent is rhizosphere competent, the disease protection period for the plant could be extended through the life of the crop in the field or the plant in the landscape (Wissuwa et al., 2009). We investigated the feasibility of using
42 dairy cow compost as a soil amendment in conjunction with the nematode Aphelenchus avenae as an alternative to chemical control for soilborne fungal pathogens and their diseases in greenhouse and transplant crop production systems.
2.1.1. Host-Pathogen Systems
Three model host-pathogen systems were used to evaluate and demonstrate the efficacy of faunal control and compost amendment, including:
A. Tomato (Lycopersicon esculentum L.) inoculated with Pythium ultimum, and
B. The bedding plants Impatiens balsamina L. and vinca (Catharanthus roseus)
attacked by Rhizoctonia solani
Tomato attacked by Pythium ultimum and disease control
Damping-off is an important disease of greenhouse tomatoes, causing important losses in nurseries where young and susceptible transplants are produced. Pythium ultimum is probably the most abundant and widespread plant pathogenic Pythium species in soil
(Pieczarka and Abawa, 1978). Currently, there are no tomato cultivars used in greenhouse crop production that are resistant to Pythium spp., including P. ultimum, a main causal agent of pre- and post-emergence damping-off in tomatoes (Gravel et al., 2005). Additionally, there are no registered chemical fungicides for use in greenhouse tomato production systems to control this pathogen. As such, disease management and control systems depend on cultural practices such as proper irrigation and steam sterilization of soil, but there is no consistently effective strategy (Paulitz, 1997). Since cultural practices usually are not enough to control
43 tomato seedling damping off by P. ultimum in greenhouse production systems, alternative management strategies are needed.
Vinca and Impatiens attacked by Rhizoctonia solani and disease control
Rhizoctonia solani overwinters as mycelium or sclerotia in the soil or in infected plant material. In addition to infecting a wide range of host plants, R. solani is commonly found in a wide range of soils. For many strains of R. solani, the optimum infection temperature range is 15-18ºC. As with many soilborne plant pathogens, R. solani is especially aggressive in wet, poor draining soil.
Control of R. solani is difficult in conducive soils due to the pathogen’s extensive host range, quick growth rate, and the ability of its survival structures to remain viable in the soil for years (Abeysinghe, 2009). Some cultural practices, such as crop rotation, are effective due to the limited host range of specific anastomosis groups. For example, since
AG-3 isolates of R. solani infect mostly potatoes, rotating to a different crop can help reduce inoculum levels (Loria et al., 1997). Other cultural practices, including providing good field drainage, and managing susceptible weeds and alternate hosts for the pathogen can also help reduce inoculum densities. Since sclerotia can survive for long periods of time once they are produced, these cultural practices may only reduce levels and not eliminate the pathogen.
Since the pathogen can be transported in infested soil and other materials and equipment, sanitary practices are also important in disease management.
44 The management of R. solani with chemicals often has not been sufficiently successful in the field due to the variety of factors (Grover and Kataria, 1985; De Curtis et al., 2010). In greenhouse production systems of specific crops, where conditions are more easily controlled than in the field, fungicides may be more consistently effective. However, few products are registered for greenhouse use, highlighting the needs for alternative management practices.
2.1.2. The Specific Objective
The specific objective of this research was to evaluate the efficacy of the nematode A. avenae as a biocontrol agent used to control pre- and post-emergence damping-off caused by
P. ultimum in tomato seedlings and pre- and post-emergence damping-off caused by R. solani in vinca and impatiens seedlings in a greenhouse production system when amended with various rates of dairy cow compost.
2.2. Methods and Materials
2.2.1. Preparation of inoculum of nematode Aphelenchus avenae and Pythium ultimum, and determinations of germination rates and pathogen inoculum rates
Tomato seeds of cultivar “Big Boy” were obtained from Wyatt-Quarles Seed
Company (Garner, NC) and vinca seeds of cultivar “Peppermint Cooler” and impatiens seeds of cultivar “Super Elfin White” were obtained from Hazzards Wholesale Seed Store
(www.hazzardsgreenhouse.com). A germination test using 100 seeds each was conducted by placing the seeds on moistened sterile P5 filter paper (9 cm diameter) (Fisher Scientific, 09-
45 801B) in a Petri dish (100x15mm, Fisher Scientific, 08-757-12). The filter paper was kept moist by adding sterilized deionized water via pipette and the Petri dish was incubated in the dark. After approximately 7 days, germination of all three cultivars reached 100%.
The population of fungal-feeding A. avenae was maintained and increased for 30 days on a non-pathogenic binucleate Rhizoctonia strain that was growing on potato dextrose agar
(PDA, Fisher Scientific) (Mikola and Setala, 1998). Nematodes were extracted overnight from the cultures for addition to the microcosms. A small plastic funnel was clamped and lined with a double layer of Kim-wipe (Kimberly-Clark Global Sales, Inc., Roswell, GA).
One culture plate with fungus, and nematodes was added to the funnel at a time, until either the funnel was full or the desired amount of plates was used. The PDA, fungi, and nematode mixture was immersed in sterilized de-ionized (DI) water and allowed to incubate overnight.
The next morning, the aqueous contents at the bottom of the funnel were collected into a beaker. The solution was then poured through a pre-cut screen with openings 1mm in size.
Once screened, the nematode population was quantified by pipetting a 5 ml aliquot into a plastic counting chamber (4x5mm). Under a microscope, a known area of the chamber was visually scanned and the nematodes counted to determine the number of nematodes per ml of suspension.
A kanamycin-tolerant isolate of P. ultimum was originally received from Dr. Craig
Rothrock at the University of Arkansas. The isolate was grown on a semi-selective medium and then transferred to 5% carrot agar (CA) (Sullivan et al., 2005) prior to inoculums preparation for the tomato trials.
46 Originally isolated from poinsettia in North Carolina, isolate RS3 (NRRL 22805) of
R. solani was used to produce inoculum for both the vinca and impatiens trials. The isolate was grown out on regular strength Potato Dextrose Agar (PDA, Difco Laboratories, Detroit,
MI) medium then hyphal tips were transferred to single cultures maintained on " strength
PDA).
For inoculum preparation, a colonized rice grain method was used (Holmes and
Benson, 1994). After preparing the autoclaved rice, cultures were established by aseptic transfer of four mycelial plugs, 3- to 4-mm in diameter from the margin of an actively growing colony of P. ultimum or R. solani. Pythium ultimum was allowed to grow on the rice grains for no more than 7 days before use. Rhizoctonia solani was allowed to colonize the rice grains for approximately 10 days before use. Daily, the culture flasks were hand-shaken by bouncing the rounded portion of the bottom against a thick mouse pad to ensure uniform colonization of the rice grains and to keep the fungus from matting the rice grains together.
Two days prior to each use, the rice grains were assayed for fungal purity and percent colonization by separately placing three individual rice grains on a plate of CA. As the rice grains were to be incorporated into the soil for infection of the seedlings, they were pulverized immediately prior to incorporation. Inoculum was prepared by pulverizing the colonized rice grains from one flask in a blender on the low setting until no whole rice grains were found intact. Then the pulverized rice grains were screened through a 2 mm sieve to remove large fragments before weighing out inoculum portions.
The pulverized inoculum of P. ultimum was added at different rates in preliminary trials to determine the amount of inoculum needed per microcosm (12 cm diameter x 10 cm
47 height plastic chambers) (Anderson and Ineson, 1982) to provide sufficient disease pressure on the host tomato plant. For the preliminary trials, five rates of pathogen inoculum were tested: 0.5g, 0.346g, 0.225g, 0.15g, and 0.0g inoculum per 450 cm# soil (potting mixture).
The pulverized inoculum was added to the soil mix in a large plastic bag and rotated for 1 minute to ensure an even dispersal of inoculum. Once filled with soil treatment mixture and pathogen inoculum, the microcosms were arranged in a randomized block design with four replications in a growth chamber in the NCSU Phytotron. The growth chamber was set to a photoperiod of 12 hours with a day temperature of 20ºC and night temperature of 16ºC. After
2 days in the growth chamber, 40 tomato seeds (Lycopersicon esculentum) of the nematode- resistant cultivar “Big Boy” (Wyatt-Quarles Seed Company, Garner, NC) were planted in each microcosm. The microcosms were watered with 50 ml of deionized water every 2nd day throughout the trial. Once seedlings began to emerge after about 5 days, stand count was taken daily for 2 weeks and the final stand counts analyzed with PC- SAS (SAS 9.1, SAS
Institute Inc., Cary, NC, USA) and significant differences declared at a p-value of 0.01. The results showed that there was no significant difference in seedling emergence among the four rates of inoculum used, but all levels were different than the control treatment. Therefore, the lowest level, 0.15g inoculum/450cm# soil, was used in all tomato trials.
Similarly, the pulverized inoculum of R. solani was added at different rates in preliminary trials to determine the amount of inoculum needed per microcosm (12 cm diameter x 10 cm height) (Anderson and Ineson, 1982) to provide sufficient disease pressure on the host vinca and impatiens plants. Five rates of pathogen inoculum were used, 0.5g,
0.346g, 0.225g, 0.15g, and the non-infested control. Four replicate pots of each compost
48 ratio (4, 8, and 16% dairy cow compost:potting soil) were established. Pots were arranged in a randomized block design in a greenhouse maintained between 25 and 20C. After a 2 day period of adjustment for the clay pots, 40 vinca seeds of cultivar “Peppermint Cooler” and 40 impatiens seeds of cultivar “Super Elfin White” were planted in each clay pot. The clay pots were watered with 50 ml of deionized water every 2nd day throughout the trial. Once seedlings began to emerge after about 6 days, stand count was taken daily for 2 weeks and the final stand counts analyzed with PC- SAS and significant differences declared at a p- value of 0.01. The results showed that there was no significant difference in the amount of vinca or impatiens seedling emergence between the three lowest rates of R. solani inoculum.
There was a significant difference in the amount of both vinca and impatiens seedling emergence between the two highest levels of inoculum and the lowest three levels. Since the highest rate of inoculum resulted in under 50% seedling emergence rate, which we considered to be too severe for our tests, all subsequent bedding plant experiments were conducted using 0.345g inoculum per pot (i.e.,450cm# soil).
For the bedding plants trials, seeds of the vinca cultivar “Peppermint Cooler” and the impatiens cultivar “Super Elfin White” were germinated in a greenhouse flat of vermiculite soilless potting mix covered by a black opaque plastic bag. The seeds were placed in the flat, lightly covered with a layer of vermiculite, gently watered, and enclosed by the plastic bag.
The flats were left undisturbed for approximately 6 days until the seeds germinated. The temperature range during germination until transplantation was 18-20ºC.
49 2.2.2. The Experimental Design, Treatments, and Data Collection
A. Experiment 1: Tomato attacked by Pythium ultimum
Experimental design
The experiment was a 4x3 factorial design with four treatments and three compost rates. Each treatment combination was replicated four times, forming a total of 48 microcosms for the tomato trial. Twelve treatment combinations (microcosms) were randomly assigned into one of four blocks (i.e., four replications), forming a randomized complete block design. There were 40 tomato seeds planted per microcosm. The experiment was conducted twice over time (Trial 1 and Trial 2).
Experimental treatments
For both the tomato trials, the four treatments were: i) nematodes plus the pathogen; ii) pathogen only (inoculated control); iii) pathogen plus a fungicide drench; and iv) no pathogen (non-inoculated control). For both trials, the rates of compost amended to the soil
(v/v) were: i) 4% dairy cow compost : 96% potting soil, ii) 8% dairy cow compost : 92% potting soil, and iii) 16% dairy cow compost : 84% potting soil. The dairy cow compost used was Daddy Pete’s Plant Pleaser (www.daddypetes.com) and the potting soil was Fafard
4P(www.fafard.com)The microcosms were blocked by replication, and arranged completely randomly with one cart in the Phytotron growth chamber serving as one block. The carts in the growth chamber were moved every 2 days in a counter-clockwise pattern a total of four times in order to minimize the effect of placement within the growth chamber.
50 Treatments were added 1 week after the microcosms were placed in the growth chamber. Once the nematodes were extracted overnight and counted, a suspension of A. avenae in water was produced containing approximately 1000 individuals per 5 ml of deionized H2O. In each microcosm receiving the nematode treatment, three individual trenches approximately 2 cm in depth were prepared using a sterilized stainless steel spatula.
Prior to pipetting 5 ml of the solution into the pre-prepared trenches in each microcosm, the nematode solution was stirred vigorously to ensure an even distribution of individuals. After pipetting, the trenches were covered with soil. For the tomato trials, the fungicide treatment was Ridomil Gold EC (47.6% mefenoxam, Novartis). The fungicide drench was prepared by adding 12.6 ml of chemical to 950 ml of deionized water and additional water was added to make the total volume to exact 1000 ml with a fungicide concentration of 0.6% product.
Then, 5 ml of fungicide solution was sprayed to the surface of each microcosm. All non- inoculated control and inoculated control microcosms received 5 ml of deionized H2O at this time.
One week after the application of the treatments, 40 tomato seeds were planted in each microcosm. Similar to the nematode suspension application, three individual trenches were dug, approximately 2.5 cm deep. The tomato seeds were distributed evenly amongst the three trenches in each microcosm and covered with soil. After covering the seeds with soil,
50 ml of deionized water were added to each microcosm. The microcosms were watered with
50 ml of deionized water every second day throughout the trial. Once tomato seedlings began to emerge after about 5 days, seedlings were counted daily for 4 weeks.
51 Data collection
At the conclusion of the tomato experiments, seedlings from each of the four replications of each of the four treatments were assayed for the presence of the pathogen.
After removing the seedlings from the microcosms, soil debris was washed from the roots and crowns. Seedling tissue was then disinfected with 1% bleach (NaOCl) for 2 minutes, washed in sterile deionized water for 1 minute and allowed to air dry on sterile filter paper
(Al-Sa’di et al., 2007). Once dry, tissue was transferred to PARPY semi-selective medium and incubated for 7 days at room temperature (Jeffers and Martin, 1986). Pythium ultimum was successfully isolated from tomato seedlings grown in nematode, fungicide, and inoculated control microcosms for all four replications, but was not isolated from seedlings grown in non-inoculated control microcosms for any of the four replications. The results were similar in both tomato trials.
In the second run of the tomato experiment, nematodes were recovered from the nematode microcosms at the end of the trial. Soil from each of the nematode treatments was washed for the recovery of A. avenae using the North Carolina Elutriator (Byrd, Jr. et al.,
1976). Each of the nematode treatment microcosms was treated as an individual sample and the samples from each replication were elutriated on the same run for a total of 4 runs. After elutriation, nematodes were counted for each sample.
For the tomato experiment, the main indicator for assessing disease activities was the seedling emergence. Data was also collected on the pathogen. The relative activity of
Pythium ultimum was calculated in each trial for each treatment by subtracting number of
52 surviving seedlings in the treatments inoculated with the pathogen (Spat) from number of surviving seedlings in the non-infested control treatment (Scon) using the following equation:
Relative activity of Pythium ultimum (%) = (Scon-Spat)/Scon * 100
Statistical analysis:
We examined results using the general linear model to test the main effects of experimental treatments and their interactions. The homogeneity test was first conducted for the data of two experimental trials to determine whether they were significantly different. If yes, data from two trials was then analyzed separately. In this tomato experiment, there was significant difference between the two trials and the two trials were therefore analyzed separately and data were then presented accordingly. All statistical analyses were performed using SAS 9.1. For all tests, P ! 0.05 was considered to indicate a statistically significant difference.
B. Experiment 2: Bedding plants attacked by Rhizoctonia solani
Experimental design
The experiment was a 4x3 factorial design with four treatments and three compost rates. Each treatment combination was replicated four times, forming a total of 48 pots for each of the vinca and impatiens trials. Twelve treatment combinations (pots) were randomly assigned into one of four blocks (i.e., four replications), forming a randomized complete
53 block design design. There were three vinca or impatiens seedlings planted per pot. The experiment was repeated twice over time (Trial 1 and Trial 2).
Experimental treatments
For the bedding plants trials, the four treatments were: i) nematodes plus the pathogen; ii) pathogen only (inoculated control); iii) pathogen plus a fungicide drench ; and iv) no pathogen (non-inoculated control). For the bedding plants trials, the rates of compost amended to the soil were: i) 4% dairy cow compost : 96% potting soil, ii) 8% dairy cow compost : 92% potting soil, and iii) 16% dairy cow compost : 84% potting soil. The pots were blocked by replication, and arranged completely randomly with one bench in the greenhouse serving as one block.
Faunal treatments were added 1 week after the pots were placed in the greenhouse.
Once the nematodes were extracted overnight and counted, a suspension of A. avenae in water was produced containing approximately 1000 individuals per 5 ml of deionized H2O.
In each pot receiving the nematode treatment, three individual trenches approximately 2 cm in depth were prepared using a sterilized stainless steel spatula. Prior to pipetting 5 ml of the solution into the pre-prepared trenches in each pot, the nematode solution was stirred vigorously to ensure an even distribution of individuals. After pipetting, the trenches were covered with soil. For the bedding plant trials, the fungicide treatment was Banrot 40 WP
(thiophanate-methyl 25% + etridiazole 15%, The Scotts Company LLC). The fungicide drench was prepared by adding 1.3g of Banrot to 950 ml of deionized water and additional water was added to make the exact volume of the solution at 1000 ml with the concentration
54 of 0.13% product. Then, 5 ml of fungicide was sprayed to the surface of each microcosm.
All non-inoculated control and inoculated control pots also received 5 ml of deionized H2O at this time.
One week after the application of the treatments, three vinca or impatiens seedlings were transplanted to each pot. Following the transplanting, 50 ml of water were added to each pot. The pots were watered with 50 ml of water every second day throughout the trial.
Once the bedding plant seedlings began to emerge approximately 3 days after transplant, seedlings were counted daily for 3 weeks. Data was also recorded on the number of emerged seedlings with disease symptoms.
Data collection
At the conclusion of the bedding plant experiments, seedlings from each of the four replications of each of the four treatments were assayed for the presence of the pathogen.
After removing the seedlings from the pots, soil debris was washed from the roots and crowns. Seedling tissue was then disinfected with 1% bleach (NaOCl) for 2 minutes, washed in sterile deionized water for 1 minute and allowed to air dry on sterile filter paper (Al-Sa’di et al., 2007). Once dry, tissue was transferred to PARPY semi-selective medium and incubated for 7 days at room temperature. Rhizoctonia solani was successfully isolated from vinca and impatiens seedlings grown in nematode, fungicide, and inoculated control clay pots for all four replications, but was not isolated from seedlings grown in non-inoculated control clay pots for any of the four replications. The results were identical for both the vinca and impatiens trials.
55 At the conclusion of both vinca and impatiens experiments, nematodes were recovered from the nematode pots. Soil from each of the nematode treatments was washed for the recovery of A. avenae using the North Carolina Elutriator (Byrd et al., 1976). Each of the nematode treatment pots was treated as an individual sample and the samples from each replication were elutriated on the same run for a total of four runs. After elutriation, nematodes were counted for each sample.
For the bedding plants experiments, the main disease symptom observed was disease severity. The data for the two trials for both vinca and impatiens was not significantly different at the date of interest and so the results are presented together (see more below).
Statistical analysis:
We examined results using the general linear model to test the main effects of experimental treatments and their interactions. The homogeneity test was first conducted for the data of two experimental trials to determine whether they were significantly different.
There was no significant difference between the two trials and data from trials were then analyzed as a repeated randomized complete block design. All statistical analyses were performed using the SAS 9.1). For all tests, P ! 0.05 was considered to indicate a statistically significant difference.
2.3. Results
In the both the first and second tomato trial, only the treatment variable was significant in terms of disease suppression; neither the compost variable nor the interaction
56 between the treatment and compost variables were significant in terms of disease suppression
(Table 2.1).
Table 2.1. ANOVA table for both tomato trials.
Experimental trial Variable Pr > F
Treatment 0.0291
Trial 1 Compost 0.2300
Trt*Comp 0.6364
Treatment <0.0001
Trial 2 Compost 0.5245
Trt*Comp 0.1362
Data shown are the results of the general linear model test. P ! 0.05 was
considered to indicate a statistically significant difference.
A. Experiment 1: Tomato attacked by Pythium ultimum
The homogeneity test showed that there were significant differences between two experimental tomato trials and the data from two trials were therefore presented separately.
57 Germination and effects of treatments
Tomato seedlings began to emerge in the first week but maximum emergence was not observed in the microcosms without P. ultimum until week four. In the first tomato trial, at week four, the pathogen + nematode treatment, the pathogen + fungicide treatment, and the no pathogen treatment were only significantly different from the pathogen only treatment control in terms of average seedling emergence but not from each other (Figure 2.1a).
In the second tomato trial at week four, however, the pathogen + nematode treatment, pathogen + fungicide treatment, and the no pathogen treatment were significantly different both from each other and the pathogen only treatment control in terms of average seedling emergence (Figure 2.1b).
The final seedling emergence was significantly different between two different trials with the average emergence rate reaching at around 70% in Trial 1 and only about 35% in
Trial 2.
58
Figure 2.1. Emergence of tomato seedlings in growth chamber microcosms: a) Trial 1 and b) Trial 2. Pathogen + nematode: Pythium ultimum and the nematode Aphelenchus avenae, Pathogen only: Pythium ultimum; Pathogen + fungicide: Pythium ultimum plus the fungicide mefenoxam; No pathogen: the no pathogen and no treatment control. Data represent final emergence at week four in both trials and are the mean percentage of 40 seeds per microcosm over four replications plus their corresponding standard errors. Columns with a different letter are significantly different at p=0.05 according to PROC GLM with the LSD means comparison.
59 Effects of compost rates
In the first tomato trial, at week four, the three compost rates ( 4%, 8%, and 16%) were not significantly different from each other in terms of average seedling emergence
(Figure 2.2a). Similar results were also observed in the second trial with no differences in the average seedling emergence among the 4%, 8%, and 16% compost rates (Figure 2.2b).
60
Figure 2.2. Germination rates of tomato seeds as influenced by the rate of compost: a) Trial 1 and b) Trial 2. Data represent final emergence at week four of each trial and are the mean percentage of 40 seeds per microcosm over four replications plus the standard errors. Columns with a different letter are significantly different at p=0.05 according to PROC GLM.
61 Pathogen activity
For both the first and second tomato trial, relative activity of P. ultimum was higher in the pathogen only control microcosms than in microcosms receiving either the nematode treatment or fungicide treatment (Table 2.2).
Table 2.2. Relative activity of Pythium ultimum in growth chamber microcosms amended with the nematode Aphelenchus avenae, drenched with the fungicide mefenoxam, or left untreated.
Experimental trial Treatment Relative activity of Pythium ultimum
Pathogen + nematode -1.50%
Trial 1 Pathogen only 10.50%
Pathogen + fungicide 0.24%
Pathogen + nematode 0.00%
Trial 2 Pathogen only 5.63%
Pathogen + fungicide 0.00%
Data represent final pathogen activity in the first tomato trial and were calculated for each treatment by subtracting number of surviving seedlings in each specific treatment inoculated with the pathogen (Spat) from number of surviving seedlings in the non-inoculated control treatment (Scon) using the following equation: Relative activity of Pythium ultimum (%) = (Scon-Spat)/Scon * 100. The data are the mean percentage of 40 seeds per microcosm over four replications.
62 Nematode populations
There was no significant difference in the average total count of nematodes extracted at the conclusion of the second tomato trial from microcosms receiving the pathogen + nematode treatment amended with the middle compost rate (8%), and from microcosms receiving the pathogen + nematode treatment amended with the high compost rate (16%).
There was a significant difference in the average total count of nematodes extracted at the conclusion of the second tomato trial from microcosms receiving the pathogen + nematode treatment amended with the low compost rate (4%), and the middle and high compost rates
(8% and 16%) (Figure 2.3).
Figure 2.3. Average number of Aphelenchus avenae extracted from microcosms receiving the pathogen + nematode treatment, broken down by compost rate (%) in the second tomato trial. Data represent average total nematode count at week 11 over four replications per compost rate. Columns with a different letter are significantly different at p=0.05 according to PROC GLM.
63 B. Experiment 2: Bedding plants attacked by Rhizoctonia solani
The homogeneity test showed that there were no significant differences between two experimental trials and the data from two trials were therefore combined. In the vinca trial at four weeks, the treatment variable, the compost variable, and the interaction between the treatment and compost variables were significant in terms of disease suppression. Also in the impatiens trial at four weeks, the treatment variable, the compost variable, and the interaction between the treatment and compost variables were significant in terms of disease suppression
(Table 2.3).
Table 2.3. ANOVA table for the bedding plants trials.
Experimental trial Variable Pr > F
Treatment <0.0001
Vinca Compost 0.0002
Trt*Comp 0.0009
Treatment <0.0001
Impatiens Compost <0.0001
Trt*Comp <0.0001
Data shown are the results of the general linear model test. P ! 0.05 was
considered to indicate a statistically significant difference.
64 Germination and effects of treatments
Vinca seedlings began to emerge in the first week but maximum emergence was not observed in the microcosms without R. solani until week four. In the vinca trials, at week four, the pathogen + nematode treatment, the pathogen + fungicide treatment, and the no pathogen treatment were only significantly different from the pathogen only treatment control in terms of disease incidence but not from each other (Figure 2.4).
Figure 2.4. Disease incidence on vinca seedlings in greenhouse pots. Pathogen + nematode: Rhizoctonia solani and the nematode Aphelenchus avenae, Pathogen only: Rhizoctonia solani; Pathogen + fungicide: Rhizoctonia solani + the fungicide thiophanate-methyl + etridiazole; No pathogen: the no pathogen and no treatment control. Data represent final disease incidence at week four and are the mean percentage of three seedlings per pot over eight replications plus their corresponding standard errors. Columns with a different letter are significantly different at p=0.05 according to PROC GLM.
65 Impatiens seedlings began to emerge in the first week but maximum emergence was not observed in the microcosms without R. solani until week four. In the impatiens trials, at week four, the pathogen + nematode treatment, the pathogen + fungicide treatment, and the no pathogen treatment were only significantly different from the pathogen only treatment control in terms of disease incidence but not from each other (Figure 2.5).
Figure 2.5. Disease incidence on impatiens seedlings in greenhouse pots. Pathogen + nematode: Rhizoctonia solani and the nematode Aphelenchus avenae, Pathogen only: Rhizoctonia solani; Pathogen + fungicide: Rhizoctonia solani + the fungicide thiophanate-methyl + etridiazole; No pathogen: the no pathogen and no treatment control. Data represent final disease incidence at week four and are the mean percentage of three seedlings per pot over eight replications plus their corresponding standard errors. Columns with a different letter are significantly different at p=0.05 according to PROC GLM.
66 Effects of compost rates
In the vinca trial at week four, the 4% compost rate and 8% compost rate were significantly different from the 16% compost rate in terms of disease incidence on seedlings
(Figure 2.6)
Figure 2.6. Disease incidence on vinca seedlings in greenhouse pots amended with 3 rates of compost; 4%, 8%, and 16%. Data represent final disease incidence at week four and are the mean percentage of three seedlings per pot over eight replications. Columns with a different letter are significantly different at p=0.05 according to PROC GLM.
In the impatiens trial, at week four, three compost rates (4%, 8%, and 16%), were significantly different from each other in terms of disease incidence on seedlings (Figure
2.7).
67
Figure 2.7. Disease incidence on impatiens seedlings in greenhouse pots amended with 3 rates of compost; 4%, 8%, and 16%. Data represent final disease incidence at week four and are the mean percentage of three seedlings per pot over eight replications. Columns with a different letter are significantly different at p=0.05 according to PROC GLM.
Nematode populations
There was no significant difference in the average total count of nematodes extracted at the conclusion of either the first or second vinca trial or the first or second impatiens trial from pots receiving the pathogen + nematode treatment amended with the middle compost rate (8%), and from microcosms receiving the pathogen + nematode treatment amended with the high compost rate (16%). There was a significant difference in the average total count of nematodes extracted at the conclusion of the first and second vinca trial and the first and second impatiens trial from pots receiving the pathogen + nematode treatment amended with the low compost rate (4%), and the middle and high compost rates (8% and 16%) (Figures
2.8 & 2.9).
68
Figure 2.8. Average number of Aphelenchus avenae extracted from pots receiving the pathogen + nematode treatment, broken down by compost rate (%) in the two vinca trials. The gray columns represent the first vinca trial and the black columns represent the second vinca trial. Data represent average total nematode count at week 6 over four replications per compost rate. Columns with a different letter are significantly different at p=0.05 according to PROC GLM.
Figure 2.9. Average number of Aphelenchus avenae extracted from pots receiving the pathogen + nematode treatment, broken down by compost rate (%) in the two impatiens trials. The gray columns represent the first impatiens trial and the black columns represent the second vinca trial. Data represent average total nematode count at week 6 over four replications per compost rate. Columns with a different letter are significantly different at p=0.05 according to PROC GLM.
69 2.4 Discussion and Conclusions Our results showed that the fungal-feeding nematode A. avenae can effectively suppressed tomato seedling damping-off caused by P. ultimum and bedding plant seedling damping-off caused by R. solani. Previous studies have reported that Pythium species can be controlled by other fungi and bacteria, but there has been no other report of control of
Pythium by nematodes (Drechsler, 1943; Al-Hamdani et al., 2003). The fungal-feeding habit of A. avenae is well-known but most studies have been focused on their contribution to N mineralization (Chen and Ferris, 1999; Chen et al., 2001; Okada and Ferris, 2001; Okada and
Kadota, 2003). In our experiment, however, nematode grazing achieved similar efficacy
(Trial 1) or even higher efficacy (Trial 2) than the standard rate of fungicide drench in suppressing tomato seedling damping-off caused by P. ultimum. This result suggests that if properly managed, the nematode A. avenae can be used as an effective alternative to mefenoxam for the control of tomato seedling damping-off caused by P. ultimum.
Similar to the nematode suppression of P. ultimum, nematodes significantly suppressed seedling damping-off caused by Rhizoctonia solani in the bedding plant trials as well (Figures 2.4 and 2.5). The nematode A. avenae has been shown to be effective in suppressing R. solani in a few other experiments (Friberg et al. 2005). On petri plates with the presence of other fungi, A. avenae has been showed with low to moderate preference of
R. solani (Okada et al., 2005; Hansa et al., 2006). Nematode population reproduction on R. solani was also higher than on other soil fungi, indicating A. avenae is able to receive adequate nutrition through hyphal grazing on the pathogen (Ishibashi et al., 2005). A. avenae seemed to be more effective in grazing R. solani and suppressing the disease when R. solani
70 was the dominant food source. For example, Lootsma and Scholte (1997) demonstrated that addition of the nematode A. avenae suppressed Rhizoctonia stem canker on potato.
Therefore, the nematode A. avenae may be a viable disease control method for suppression of bedding plant seedling post-emergence damping-off caused by Rhizoctonia solani. This could be an important area for future research as R. solani has a large host range and large distribution range. Though fungicides are capable of effective suppression of R. solani and its diseases, mycophagous nematodes can be an effective alternative to chemical control.
It has been reported that the quantity and quality of organic inputs can critically affect the disease suppression efficiency in soil (Tamm et al., 2010). In our study, disease suppression was not affected by the percentage of compost amended to the soil in the tomato trials. This result was in keeping with the results of other researchers indicating multiple compost blends capable of Pythium disease suppressive abilities (Scheuerell et al., 2004).
However, disease suppression was significantly affected by the percentage of compost amended to the soil in the bedding plant trials with disease suppression being greatest in seedlings receiving the highest percentage (16%) of compost. This is in keeping with previous results that pre-emergence impatiens seedling damping-off was lower in composted swine waste-amended potting soil than non-amended potting soil (Diab et al., 2003). Higher percentage of compost likely increased microbial growth, thereby enhancing the direct microbial antagonists to pathogens as well as the food sources for fungal-feeding nematodes.
It should be mentioned that the emergence rates between the two tomato trials were significantly different (Figures 2.1 a, b). We speculate that this difference stemmed from the temperature difference caused by a failure of the incandescent lights in the growth chamber
71 during Trial 2. There the incandescent lights failed for about 8 days before repair and our previous test found that this could result in ca. 3°C decrease of temperature in the very surface layer of the soil in microcosms. Lower temperature may increase P. ultimum activities as well as reduce the vigor of germinating tomato seeds. It is also interesting to note that the relative efficiency of nematode suppression of P. ultimum remained unchanged as evidenced by seedling emergence in each trial. This result suggests that nematodes may be effective in different soil temperature regimes.
Significantly different numbers of nematodes re-isolated from the three levels of compost suggest that the proportion of compost amendment in the soil treatment had a significant effect on the survival and/or reproduction of the nematodes in the soil. The underlying mechanisms for this trend are unknown. It is well documented that additions of composts and other organic manures to soil increase the populations of microbial-feeding mesofauna (Scholte and Lootsma 1998; Forge et al., 2003). Since peat-moss is generally inactive and provides little energy and nutrient sources for microbes, it is reasonable to expect that the higher compost favors fungal growth and thus provides a more stable food source for fungal-feeding mesofauna. Previous research has indicated that the incorporation of organic residues, such as compost, may result in the increase of microbial biomass carbon and growth (Mukherjee et al., 1990; Tu et al., 2006; Tejada et al., 2008). One study conducted on different compost types found that a cattle manure compost had the highest levels of organic C and total N compared to the other compost types. Additionally, the cattle manure compost had the highest level of microbial colonization (Gattinger et al., 2004). It is plausible to postulate similar microbial biomass and colonization levels in our experiments as
72 the compost amendment used as a soil treatment in our experiments was formed from dairy cow manure. Other factors may also contribute to the observed differences in nematode population densities between compost percentages of soil amendments. For example, higher compost percentages in the soil amendments may allow more water to be absorbed and retained, thereby creating a more stable and supportive environment for the nematode population’s growth and development.
The highest population of nematodes in both the tomato and bedding plant trials was extracted from the 16% compost soil treatment. The proportion of compost in the soil not only affected the survival and reproduction of the nematodes but may also have enhanced their disease suppression ability. Though all our soil treatments contained a percentage of compost amendment, our bedding plant trials’ results suggest that disease suppression is increased with compost amendment. However, the average total count of nematodes ultimately re-isolated from the amended soil did increase as the percentage of compost amended increased. These results suggest that though nematode density at 4% compost amendment may be sufficient to suppress disease under some given conditions, higher percentages of compost amendment support greater population numbers of nematodes, ensuring a high predation pressure on fungal pathogens, particularly in environments with high fluctuations.
In conclusion, our experiments showed that the nematode A. avenae was capable of providing a level of disease suppression comparable to the standard fungicide. The fungal- feeding nematode was better supported as the percentage of incorporated compost increased, but this linear relationship did not correlate with more disease suppression on tomato
73 seedlings, but did occur in the experiment with the bedding plants. As non-chemical disease management strategies for greenhouse use, both mycophagous nematodes and dairy cow compost amendments show promise. Their conjunctive use may be an avenue worth exploring further for incorporation into integrated pest management practices for vegetable growers and bedding plant producers looking for an eco-friendly option of suppressing
Pythium ultimum and Rhizoctonia solani seedling damping-off.
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79 Chapter 3. The nematode Aphelenchus avenae and mycophagous
collembolan and mites as control methods for bell pepper seedling
damping-off caused by Phytophthora capsici
3.1 Introduction
Phytophthora species cause a wide range of diseases on an equally wide range of host plants. Most Phytophthora species cause root rots, damping-off, and necrosis of stems, tubers, and corms. However, some Phytophthora species can also cause bud and fruit rots, and foliage, twig, and fruit blights. Phytophthora species causing root and stem rot occur primarily in moist environments in warm climates (Agrios, 2005). This soilborne pathogen is usually polycyclic, survives in host tissues or as dormant propagules, and can reproduce both sexually and asexually (Bonnet et al., 2007).
Phytophthora capsici produces three spore types for dissemination, survival, and germination. Water, including droplets that splash onto surfaces, is the means of dissemination for sporangia, which can then either germinate directly or release swimming zoospores. Sporangia are produced on multiple plant parts, including roots, crowns, and fruit.
In greenhouse situations, both sporangia and zoospores can be transported via irrigation and drainage water, and in the field can also be transported via rain and wind (Hausbeck and
Lamour, 2004). When soils become saturated with water, dormant oospores are stimulated to germinate to produce the sporangia. In dry conditions, oospores can remain dormant outside
80 of host tissue in the soil for years due to their melanized thick walls (Erwin and Ribeiro,
1996).
3.1.1. Bell pepper attacked by Phytopthora capsici and disease control
Phytophthora capsici causes seedling damping-off in bell pepper plants, and is a major threat to bell pepper production worldwide (Babadoost, 2004). Historically, fungicide use has been heavily relied upon for the control of Phytophthora spp. The phenylamide fungicides, including metalaxyl and mefenoxam, have been used by many growers to control
P.capsici. Both fungicides have been found to be highly effective against sensitive P. capsici isolates, however, adaptation to this class of fungicides is common in oomycetes and considered to be an inevitable occurrence due to the specificity of this class of fungicides
(Davidse et al., 1991). Regardless, few fungicides are registered for use in greenhouse production. Though crop rotation is an important cultural practice for disease management, it is not a stand-alone strategy due to the long-term survival of oospores in the soil, and it is not relevant in greenhouse situations. Since P. capsici zoospores are transported via water, the inoculum can be easily spread throughout greenhouse production houses by recirculating irrigation water, causing epidemics. Because damping-off of pepper seedlings caused by
P.capsici is difficult to control using chemical and cultural management strategies, biological control methods should be studied and developed for additional disease control efficacy.
81 3.1.2. The Specific Objective
The specific objective of this research was to evaluate the efficacy of the nematode
Aphelenchus avenae, a mycophagous collembolan and mite as biocontrol agents used to control post-emergence damping-off caused by Phytophthora capsici in bell pepper seedlings growing in soil when amended with various rates of dairy cow compost.
3.2. Methods and Materials
3.2.1. Preparation of nematode A. avenae, mycophagous collembolan and mites, and pathogen Phytophthora capsici, and determinations of germination rates and pathogen inoculum rates
Bell pepper seeds of cultivar “Camelot” were obtained from Wyatt-Quarles Seed
Company (Garner, NC). A germination test using 100 seeds was conducted by placing the seeds on moistened P5 filter paper with a diameter of 9cm (Fisher Scientific, 09-801B) in a
Petri dish (100x15mm, Fisher Scientific, 08-757-12). The filter paper was kept moist using sterilized deionized water via pipette and the Petri dish was incubated in the dark. After approximately 7 days, germination reached 100%.
The population of A. avenae was maintained and increased on a non-pathogenic binucleate Rhizoctonia strain grown on potato dextrose agar (PDA) (Fisher Scientific) for 30 days (Mikola and Setala, 1998). The nematodes reproduced rapidly and produced large populations within a month. Nematodes were extracted overnight from the cultures for addition to the microcosms. A small plastic funnel was clamped and lined with a double layer
82 of Kim-wipe (Kimberly-Clark Global Sales, Inc., Roswell, GA). One culture plate with fungus, and nematodes was added to the funnel at a time, until either the funnel was full or the desired amount of plates was used. The PDA, fungi, and nematode mixture was immersed in sterilized de-ionized (DI) water and allowed to incubate overnight. The next morning, the aqueous contents at the bottom of the funnel were collected into a beaker. The solution was then poured through a pre-cut screen with openings 1mm in size. Once screened, the nematode population was quantified by pipetting a 5 ml aliquot into a plastic counting chamber (4x5mm). Under a microscope, a known area of the chamber was visually scanned and the nematodes counted to determine the number of nematodes per ml of suspension.
Both the collembola and the mite were originally isolated from vermicompost from the NCSU Department of Entomology lab of Dr. Yasmin Cardoza. Once isolated from the vermicompost, the collembola and mite populations were maintained and increased on a non- pathogenic binucleate Rhizoctonia strain growing on potato dextrose agar (Mikola and
Setala, 1998). Weekly, the plates were examined under the microscope and individuals were transferred to a new plate of PDA with the non-pathogenic binucleate Rhizoctonia strain from the old plate in groups of 10.
Soil was collected from six arbitrarily -selected sites from a bell pepper field located in eastern North Carolina with a documented history of P. capsici. Soil was collected from three different rows of pepper plants exhibiting symptoms of Phytophthora blight as well as from three non-symptomatic rows of pepper plants. The soil samples from each site were
83 combined into large coolers and transported back to the lab where they were kept at room temperature until use.
Preliminary tests of the soil collected from the field were conducted to verify P. capsici presence and density in the infested samples, and the absence of pathogen in the control soil. Eight microcosms were each filled with 450g of field soil; four with infested soil and four with control soil. Two days later, 40 pepper seeds were planted in each microcosm in three individual trenches approximately 2.5 cm deep. The pepper seeds were pre-counted and distributed evenly amongst the three trenches in each microcosm and covered with soil.
After covering with soil, 50 ml of deionized water were added to each microcosm. The microcosms were placed in a growth chamber in the NCSU Phytotron. The growth chamber was set to a photoperiod of 12 hours with a day temperature of 26ºC and night temperature of
22ºC. Throughout the duration of the test microcosms received 50 ml of deionized water every 2nd day. Once seedlings began to emerge, approximately 4 days after planting, the seedlings were counted daily. After 3 weeks, the test was concluded and data were analyzed.
Representative seedlings from both infested soil microcosms and control microcosms were assayed for the presence of P. capsici. After removing the seedlings from the microcosms, soil debris was washed from the roots and crowns. Seedling tissue was then disinfected with 1% bleach (NaOCl) for 2 minutes, washed in sterile deionized water for 1 minute and allowed to air dry on sterile filter paper (Al-Sa’di et al., 2007). Once dry, tissue was transferred to PARP semi-selective medium and incubated for 7 days at room temperature (Jeffers and Martin, 1986). Phytophthora capsici was successfully isolated from
84 the seedlings removed from the infested soil microcosms, but not from the seedlings removed from the control microcosms.
However, as the average seedling emergence rates for the seedlings grown in the control microcosms was lower than expected, the soil was steam pasteurized in greenhouse carts for 1 hour two days before filling the microcosms. Also, the average seedling emergence rates for the seedlings grown in the infested microcosms were higher than expected, so cultures of Phytophthora capsici originally isolated from the infested field soil were added to the infested soil microcosms to increase disease pressure.
The P. capsici isolate used to infest the field soil was grown on PARP and then transferred to 5% carrot agar (CA) (Sullivan et al., 2005; Kannwischer and Mitchell, 1981).
For inoculum preparation, each plate of CA was divided into squares approximately
5mm per side with a stainless steel scalpel that was flame sterilized with EtOH between plates. This was done immediately prior to adding 5 squares of inoculated agar into each microcosm of infested field soil to increase disease pressure. As this was a transplant to field soil experiment, transplant trays with 24 wells holding 15g of mix were filled with a dairy compost plus potting soil mixture at one of three rates: 4, 8, or 16% dairy compost.
3.3. The Experimental Design, Treatments, Data Collection and Analysis
3.3.1. Experimental design
The experiment consisted of two stages. Transplant seedlings were grown in the growth chamber in the first stage as described below. A split plot design was used with five
85 treatments and three compost rates for a total of fifteen treatment combinations. Once seedlings were large enough for transplanting, they were transplanted into microcosms that had been established with two types of field soil as described below. The fifteen treatment combinations were replicated eight times in each type of field soil for a total of 240 microcosms. The microcosms were randomly assigned into one of eight blocks, forming a randomized complete block design. There were two pepper seedlings transplanted per microcosm.
3.3.2. Experimental Treatments
The treatments were: i) nematodes; ii) no treatment control; iii) fungicide drench; iv) collembola; and v) mites. The rates of compost amended to the soil were: i) 4% dairy cow compost : 96%potting soil ii) 8% dairy cow compost : 92% potting soil, and iii) 16% dairy cow compost : 84% potting soil. The field soil types were i) infested field soil amended with five 5mm squares of P. capsici-colonized CA media, and ii) control field soil steam pasteurized. The commercial pepper cultivar “Camelot” was used which is highly susceptible to P. capsici. The microcosms were blocked by replication, and arranged completely randomly with one and a half carts in the growth chamber serving as one block.
One flat containing 72 plug wells with a well volume of 18 cm3 was set up for each faunal treatment for a total of five flats and 360 plug wells. Each flat was divided into thirds, with each third corresponding to each soil treatment; 4, 8, and 16% dairy cow compost
(Daddy Pete’s Plant Pleaser; www.daddypetes.com): potting soil (Fafard 4P; www.fafard.com). Once filled with the compost-amended soil, the flats were placed in the
86 growth chamber in the NCSU Phytotron, lightly watered with deionized H2O, and then covered with a transparent plastic lid. The growth chamber was set to a photoperiod of 12 hours with a day time temperature of 26ºC and night time temperature of 22ºC. The flats were allowed to stabilize for 1 week in the growth chamber before the faunal treatments were applied, with water applied as needed.
Once the nematodes were extracted overnight and counted, a suspension of A. avenae in water was produced containing approximately 500 individuals per 5 ml of deionized H2O. In each plug well receiving the nematode faunal treatment, three individual trenches approximately 2 cm in depth were prepared using a sterilized stainless steel spatula.
Prior to pipetting 5 ml of the solution into the pre-prepared trenches in each microcosm, the nematode solution was stirred vigorously to ensure an even distribution of individuals. After pipetting, the trenches were covered with soil.
The collembolans and mites were pipetted into a plastic collection vial using rubber tubing fitted with a filter. The mesofauna were pipetted in increments of 50 and 10 respectively. Once the desired amount of collembolans and mites had been collected in the vial, it was emptied directly onto the surface of the mix in into the corresponding plug well.
Since both collembolans and mites prefer darkness, they summarily burrowed down into the soil.
One week after the nematodes, collembolans, and mite faunal treatments were applied to the flats, 4 pepper seeds of cultivar “Camelot” were planted in each plug well. Similar to the nematode suspension application, two individual trenches were prepared. The pepper
87 seeds were distributed evenly amongst the two trenches in each plug well and covered with the trench soil. After covering with soil, 25 ml of deionized water were added to each plug well. The plug wells were watered with 25 ml of deionized water every 2nd day until transplant to the field soil microcosms.
As seedlings were grown for transplanting, eight replicate microcosms were established in the growth chamber by filling clay pots with 450 g of field soil for each faunal treatment and soil treatment for both types of field soil for a total of 15 treatments and 240 microcosms. Half of the total microcosms, or 120 microcosms, were filled with control field soil. The other 120 microcosms were filled with infested field soil. Each microcosm of infested field soil received 5 individual 5 mm squares of media colonized by P. capsici to increase the disease pressure on the pepper seedlings. The microcosms were placed in the
NCSU Phytotron and allowed to adjust to the growth chamber conditions while the seedlings grew in the flats.
One week after the seedlings began emerging, they were thinned from four to two seedlings per plug well. The next day, the two seedlings were transferred, along with the soil and faunal treatment in their plug well, to their correspondingly labeled microcosm. The extra seedlings were kept in their original flats in case they were needed. The microcosms were arranged in a randomized block design, with each cart and a half serving as one block.
Two days after transplanting, the fungicide treatment was applied. The fungicide treatment was Ridomil Gold EC (47.6% mefenoxam, Syngenta). The fungicide drench was prepared by adding 12.6 ml of chemical to 950 ml of deionized water and additional water
88 was added to make the total volume to exact 1000 ml and the concentration at 0.6% product.
Then, 5 ml of fungicide solution was sprayed to the surface of each microcosm. All microcosms, including those receiving the fungicide drench, received 50 ml of deionized
H2O at this time.
3.3.3. Data Collection
After transplanting, the seedlings were examined weekly and damping-off symptoms data were recorded based on diseases incidence and disease severity. Disease incidence was recorded weekly by taking the number of pepper seedlings in each microcosm showing disease symptoms with possible values of 0, 1, or 2. Data was averaged across the two seedlings per eight replications. This data collection was performed in order to chart disease progression through time for the infested field soil microcosms. Seedling symptom presence was also averaged across the eight replications and presented in graphical form by faunal treatment only and by soil treatment only. An analysis of variance was also performed on the data collected in the pepper trial for both infested field soil and control field soil using SAS to test the differences between means of the variables.
Disease severity was evaluated according to the rating scheme outlined by Ristaino
(Ristaino, 1990) using the following scale: 0 = no symptoms, 1 = wilting of the seedling with no stem lesion, 2 = wilting of the seedling and stem lesion with no girdling, 3 = girdled seedling stem, and 4 = dead seedling. Disease severity is presented with data shown for all microcosms showing disease symptoms. Disease severity as a function of time for treatment
89 combinations where symptoms were present is also shown; data is averaged across all reps within a faunal treatment + soil treatment combination.
Data was also calculated based on a pathogen activity index. The relative activity of
Phytophthora capsici was calculated in the pepper trial for each faunal treatment by subtracting number of surviving seedlings in the faunal treatments transplanted to infested field soil (Spat) from number of surviving seedlings in the non-infested field soil control treatment (Scon) divided by the number of surviving seedlings in the non-infested field soil control treatment (Scon) using the following equation:
Relative activity of Phytophthora capsici (%) = (Scon-Spat)/Scon * 100)
Thus, a microcsom with no surviving seedlings would have a pathogen activity index of 100.
Additionally, representative seedlings from each faunal treatment and soil treatment in both infested field and control field soil were assayed for isolation of P. capsici at the conclusion of the bell pepper trial. Phytophthora capsici was successfully isolated from seedlings grown in nematode, fungicide, collembolan, mite, and no treatment control microcosms for all eight reps in the infested field soil, but was not isolated from seedlings grown in any faunal treatment in the control field soil for any of the eight reps.
The microcosms receiving the nematode faunal treatment in both infested field and control field soil were elutriated at the conclusion of the experiment for the recovery of
Aphelenchus avenae individuals in the North Carolina elutriator (Byrd, Jr. et al., 1976). Each of the nematode treatment microcosms was treated as an individual sample and the samples
90 from each rep were elutriated on the same run for a total of two runs per rep per soil treatment. After elutriation, nematodes were counted for each sample.
3.3.4. Statistical analysis:
We examined results using the general linear model to test the main effects of experimental treatments and their interactions. Data from the pepper trial were then analyzed as a repeated randomized complete block design. All statistical analyses were performed using the SAS 9.1 (SAS Institute, Inc., Cary, NC, USA). For all tests, P ! 0.05 was considered to indicate a statistically significant difference.
3.4. Results
Results presented thereafter mainly focused on mesofaunal effects on pathogen activities in the infested field soil, though the same data collection methods were used for both the infested field and the control field soil microcosms.
In the pepper trial in the infested field soil microcosms, the treatment variable, the compost variable, and the interaction between the treatment and compost variables were significant in terms of disease suppression (Table 3.1).
91
Table 3.1. ANOVA table for pepper seedlings transplanted to infested field soil.
Variable Pr > F Treatment <0.0001 Compost <0.0001 Trt*Comp <0.0001
Data shown are the results of the general linear model test. P ! 0.05 was
considered to indicate a statistically significant difference.
3.4.1. Disease incidence and effects of treatments
Disease symptoms began to appear on pepper seedlings transplanted to infested field soil in the no treatment microcosms in the first week. However, disease symptoms did not appear on pepper seedlings in any of the microcosms receiving a faunal treatment until week
3, and disease symptoms never appeared on pepper seedlings in any microcosms receiving the standard fungicide drench (Table 3.2).
92
Table 3.2. Average percentage of pepper seedlings with symptoms per week following transplant to infested field soil.
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There was no significant difference in percentage of seedlings with damping-off symptoms amongst the four treatments after 5 weeks, but there was a significant difference in
93 percentage of seedlings with damping-off symptoms between the no treatment control and the four treatments. The three faunal treatments (nematode, collembolan, and mite) provided levels of disease suppression comparable to the standard fungicide drench (Figure 3.1).
Figure 3.1. Damping-off disease incidence on pepper seedlings in infested field soil microcosms as affected by treatment. Nematode: Aphelenchus avenae; No treatment; Fungicide: mefenoxam; Collembolan; Mite. Data represent final emergence at week 5 and are the mean percentage of 2 seedlings per microcosm over eight replications plus their corresponding standard errors. Columns with a different letter are significantly different at p=0.05 according to PROC GLM.
3.4.2. Effects of compost rate
There was no significant difference in percentage of seedlings with damping-off symptoms between the 8% and 16% compost rates, though there was a significant difference
94 in percentage of seedlings with damping-off symptoms between the 4% compost rate and the two higher compost rates (Figure 3.2).
Figure 3.2. Damping-off disease incidence on pepper seedlings in infested field soil microcosms as affected by the rate of compost. Data represent final emergence at week 5 and are the mean percentage of 2 seedlings per microcosm over eight replications plus their corresponding standard errors. Columns with a different letter are significantly different at p=0.05 according to PROC GLM.
3.4.3. Disease severity
Disease severity progressed the most quickly and was the most severe for the no treatment control with 4% dairy cow compost:potting soil treatment combination. The treatment combination with the slowest and least severe disease progression was shared by the nematode and collembolan faunal treatments at the 4% dairy cow compost:potting soil treatment combination (Figure 3.3).
95
Figure 3.3. Disease symptom severity as a function of time for treatment and compost rate combinations where pepper seedling damping-off symptoms caused by Phytophthora capsici appeared in infested field soil microcosms. In the legend, the first number represents compost rate and the letter represents the treatment; A = nematode, B = no treatment, C = fungicide, D = collembolan, E = mites. If there is no number following the letter, it is an average across the eight replications for that compost rate and treatment combination, else the number following the letter indicates the number of replications in the mean data point.
3.4.4. Pathogen activity
Since damping-off symptoms only progressed to the highest level (4 = death) in the no treatment control, relative activity of the pathogen could only be calculated for seedlings transplanted to infested field soil microcosms receiving no treatment. Though the highest pathogen activity was in Rep 3 at 50%, the average across all eight replications was only
35.42% (Table 3.3).
96
Table 3.3. Relative activity of Phytophthora capsici for pepper seedlings transplanted to infested field soil in microcosms amended with the nematode Aphelenchus avenae, drenched with the fungicide mefenoxam, mycophagous collembolan or mites or left untreated.
Treatment Relative activity of Phytophthora capsici
Pathogen + nematode 0.00%
Pathogen only 35.42%
Pathogen + fungicide 0.00%
Pathogen + collembolan 0.00%
Pathogen + mite 0.00%
Data represent final pathogen activity (see text for equation). The data are the mean percentage of two seedlings per microcosm over eight replications.
3.4.5. Nematode populations
The highest average number of A.avenae individuals was re-isolated from microcosms receiving the 16% compost amendment soil treatment prior to transplantation for both infested field soil and control field soil (Figure 3.4a,b). However, there was no significant difference in the average number of nematodes recovered from microcosms receiving the 8% and 16% compost amendment soil treatment prior to transplantation to the infested field soil, though the average number of nematodes recovered was significantly lower in microcosms receiving 4% than those receiving 8% or 16% compost amendment
(Figure 3.4a). In the control field soil, there was a significant difference in the average
97 number of nematodes re-isolated from each of the three rates of compost amendment (Figure
3.4b).
Figure 3.4. Average number of Aphelenchus avenae extracted from microcosms receiving the nematode treatment, broken down by compost rate (%): a) Infested Field Soil and b) Control Field Soil. Data represent average total nematode count at week 5 over eight replications per compost rate. Columns with a different letter are significantly different at p=0.05 according to PROC GLM.
98 3.5 Discussion and Conclusions
In this study, our results showed that the fungal-feeding nematode, a collembolan, and a mite significantly delayed damping-off disease symptom emergence and incidence on bell pepper seedlings compared to the infested control. There has been no other published report of fungal-feeding mesofauna as disease suppressors of the soilborne pathogen P. capsici. However, previous work conducted in our lab showed that two fungal-feeding collembolans, Hypogastrura perplexa and Sinella curviseta, as well as the nematode A. avenae, reduced the population of P. capsici by 50-85% compared to the no-fauna control in organic soils (Qi, et al., unpublished). Their experiment was also a microcosm study, but focused on soil type rather than compost amendment rate as a variable of interest.
Collembola effects on other pathogens have been reported (Sabatini and Innocenti, 2000;
Shirashi et al., 2003). For example, the fungal-feeding collembola Folsomia hidakana
Uchida & Tamura successfully suppressed cabbage seedling damping-off caused by
Rhizoctonia solani under greenhouse conditions (Shirashi et al., 2003). Mites have also been reported to suppress plant pathogens through fungal grazing (Enami and Nakamura, 1996;
English-Loeb et al., 2007). The fungal-feeding mite Scheloribates azumaensis also suppressed the soilborne pathogen R. solani and root rot of radish in greenhouse pots (Enami and Nakamura, 1996). Both the collembolan and mite were isolated from a vermicompost and there are high densities of these fauna in several vermicomposts (Yasmin Cardoza, unpublished; Sampedro and Dominguez, 2008). Together, these results suggest that fungal- feeding animals likely significantly contribute to the suppressive functions of vermicompost to soilborne pathogens observed in other experiments (Szczech, 2008; Ersahin et al., 2009).
99 Researchers found that vermicompost produced from agricultural wastes, including cattle manure, effectively suppressed cucumber seedling damping-off caused by Rhizoctonia solani
Kuhn (Ersahin et al., 2009).
Seedling damping-off caused by P. capsici was also significantly affected by the rate of compost amendment with the least amount of disease symptoms occurring in microcosms amended with the highest rate (16%) of compost amendment. Different compost rates can affect both fungal-feedling animals as well as other antagonistic organisms. In our experiment, higher numbers of nematodes were recovered in microcosms with higher compost rates (Figure 3.4). Also, fewer nematodes were recovered from the Control field soil than from the Infested field soil, which further suggests that microbial biomass was important in supporting the populations of microbial-feeding mesofauna. Other researchers have demonstrated other mechanisms through which P. capsici disease control was affected by soil amendments. For example, chitin-amended compost suppressed disease activity of the soilborne pathogen P. capsici on pepper in greenhouse tests to a level greater than in peppers grown in control compost-amended pots. The mechanism of disease suppression was determined to be through the enhancement of chitinase-producing native bacteria and their soil enzyme activities (Chae et al., 2006). Another group of researchers have recently shown that extracts from compost water are capable of suppressing disease caused by P. capsici in multiple host plants. A heat-stable chemical or chemicals present in water extracts from commercial composting facilities reduced disease incidence and severity by both root and foliar infections in pepper plants (Sang et al., 2010).
100 In addition to treatment and compost rate amendment significantly affecting disease suppression, our results indicated a significant interaction between the two variables.
Therefore, damping-off of pepper seedlings may be reduced by a combination of pest management strategies. Combining biocontrol agents may provide better soilborne pathogen control than application of a single biocontrol agent, especially when the pathogen is capable of infecting a wide range of susceptible hosts (Spadaro and Guillino, 2005). This premise has also been concluded by other researchers seeking an integrated system of soilborne pathogen and disease management. Two separate microbial organisms, one fungus and one bacteria, reduced galling of tomato seedlings by the root-knot nematode Meloidogyne incognita equally well when applied individually and when applied in combination (Gautum et al.,
2005). However, the reduction in the population of the soilborne pathogen was significantly increased when Paecilomyces lilacinus and Bacillus subtilus were both present when compared to the presence of either antagonist individually (Gautum et al., 2005). A group of researchers in Egypt recently introduced a new approach to combining biocontrol agents for an integrated approach to soilborne pathogen control by testing a range of biocontrol agents
(BCAs) and resistance inducers (RIs) to suppress cotton root rot disease. They concluded that cotton root rot disease was effectively suppressed via multiple mechanisms including the activation of plant systemic resistance by RIs and the targeted suppression of the pathogen by compatible BCAs (Abo-Elyousr et al., 2009).
Our results that both treatments and compost amendments applied prior to seed planting in the greenhouse may provide disease suppression may have important implications. Since the seedling stage and the time immediately following field transplant are
101 the most susceptible phases of plants to soilborne pathogens such as P. capsici, enhancement of pathogen/disease suppression in the field through pre-transplant actions could be especially important (Kim et al., 1989). Field tests are needed to examine the applicability of these fungal-feeding animals for disease control.
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