Biological Control of Phytopathogenic Fungi II

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Biological Control of Phytopathogenic Fungi (II)

• Fundamentals of Biological Control
• Fall 2005

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Last Lecture

• Plant Pathogenic Fungi
• Biological agents of PP fungi
• Bacteria
• Fungi
• Mycoviruses
• Mechanisms of Antagonism
• Antibiosis
• Competition (Direct and Cross Protection)
• Parasitism (Hyperparasitism and Hypovirulence)
• Induced resistance
• Bioactive metabolites

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Goals for Today

• Biologicals for fungal disease management
• Overview
• Specific Examples

McSpadden, B., and D. Fravel. 2002. Biological
control of plant pathogens Research,
commercialization and application in the USA.
APS Net. (http//www.apsnet.org/online/feature/bi
ocontrol/top.html)
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Mycoparasite Products
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Mycoparasite Products
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Mycoparasite Products
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Mycoparasite Products
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Mycoparasite Products
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Mycoparasite Products
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Ampelomyces quisqualis

• AQ10 Biofungicide
• Biocontrol Organism Ampelomyces quisqualis
  isolate M-10
• Target Pathogen/Disease powdery mildew
• Crop apples, cucurbits, grapes, ornamentals,
  strawberries, tomatoes
• Formulation water-dispersible granule
• Application Method spray
• Manufacturer/Distributor Ecogen, Inc., 2005
  Cabot Blvd. West, Langhorne, PA 19074 Phone
  1-215-757-1590 FAX [phone] or P. O. Box
  4309, Jerusalem, Israel Phone [phone]
  FAX [phone]

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Ampelomyces quisqualis

• Hyperparasite of powdery mildews
• Powdery mildews 256 plant species within 172
  genera in 59 families
• Colonizes hyphae, conidiophores, cleistothecia
• Direct penetration
• Host cells are killed shortly after pycnidial
  formation (2-4 days after infection)

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Ampelomyces quisqualis

• Attacks and has the potential to kill all powdery
  mildews
• Acceptable control can be obtained in greenhouse
  and field-grown vegetables.
• Repeated application, high humidity, and rainfall
  are needed
• Efficacy can vary since the host is needed for
  reproduction

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Coniothyrium minitans

• Biocontrol Organism Coniothyrium minitans
• Target Pathogen/Disease Sclerotinia sclerotiorum
  and S. minor
• Crop canola, sunflower, peanut, soybean, and
  vegetables (lettuce, bean, tomato)
• Formulation water dispersible granule
• Application Method spray
• Manufacturer/Distributor
• CONTANS Prophyta Biologischer Pflanzenschutz
  GmbH, Inselstrasse 12, D-23999 Malchow/Poel,
  Germany Phone [phone] FAX [phone]
• KONI BIOVED, Ltd., Ady Endre u. 10, 2310
  Szigetszentmiklos, Hungary Phone [phone]
  email boh8457_at_helka.iif.hu

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Sclerotinia sclerotiorum

• Sclerotinia sclerotiorum is among the most
  nonspecific, omnivorous, and successful of plant
  pathogens. Plants susceptible to this pathogen
  encompass 64 families, 225 genera, and 361
  species (Purdy, 1979). Some hosts are cabbage,
  common bean, citrus, celery, coriander, melon,
  squash, soybean, tomato, lettuce, and cucumber

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Coniothyrium minitans

• Campbell, W. A., 1947 - first observation
• Mycelial effects
• Mechanical penetration
• Endo and exo ß-1,3 glucanases, chitinase
• Intracellular colonization
• Sclerotial effects
• Mechanical and enzymatic penetration
• Colonization, internal cell collapse
• Pycnidia formation 14 days

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Coniothyrium minitans will colonize the sclerotia
of Sclerotinia sclerotiorum. An infected
sclerotium will have hundreds of pycnidia on the
surface.
Each pycnidium will release thousands of conidia
spores.
From http//www.ncsrp.com/whitemold/bioconwm.htm
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C. minitans

• Coniothyrium minitans treated soils become
  suppressive
• Decreased wilt from 90 to 10 in a 7 year
  monocrop of sunflower (control field)
• Same trend in fields treated with C. minitans,
  Trichoderma and Gliocladium
• Addition of sclerotia did not increase disease

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Fusarium oxysporum
Fusarium oxysporum is an anamorphic fungus that
contains 25 forma specialis (f. sp.) pathogenic
variants, and many nonpathogenic forms.
Fusarium oxysporum has been studied primarily
because of its pathogenic ability on plants.
Left to right, Fusarium wilt of tomato, Panama
wilt, and head scab of wheat.
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Fusarium oxysporum
Despite interest in pathogenic forms, its
distribution and ecological activities suggest a
more diverse repertoire. It is commonly isolated
from asymptomatic roots of plants, capable of
aggressively colonizing plant residue, and
rapidly reoccupy fumigated soils. Thus, it has a
remarkable persistence without requiring a
pathogenic mode of nutrition.
http//www.doctorfungus.com/thefungi/fusarium.htm
Fusarium oxysporum is produces banana-shaped,
multicelled macroconidia (above), microconidia,
and chlamydospores. It should be noted that
substantial populations of F. oxysporum are found
in native plant communities and in no instance is
it pathogenic.
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Trichoderma

• Biocontrol Agent Trichoderma harzianum Rifai
  strain KRL-AG2 (T-22)
• Target Pathogen/Disease Pythium spp.,
  Rhizoctonia solani, Fusarium spp.
• Crop trees, shrubs, transplants, all
  ornamentals, cabbage, tomato, cucumber
• Formulation granules or wettable powder
• Application granules mixed with soil or potting
  medium powder mixed with water and added as a
  soil drench
• Manufacturer/Distributor Bioworks, Inc., 122
  North Genesee St., Geneva, NY 14456 USA Phone
  1-315-781-1703 FAX [phone]

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Trichoderma spp.

• Species of Trichoderma are common in soil
  (especially water-logged soil), dung, and
  decaying plant materials
• Fast-growing, white, green, or yellow sporulating
  filaments. Conidiophores produce side branches
  bearing whorls of short phialides. Single-celled
  conidia produced successively from the tips of
  the phialides and collect in small wet masses.
• Trichoderma species are strongly antagonistic to
  other fungi. Appears to kill other fungi with a
  toxin and lytic enzymes. Can be serious pests in
  cultivated mushroom beds.

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Trichoderma spp.

• Many species of Trichoderma have been studied,
  several developed into products
• Temperature
• T. polysporum and T. viride like cool conditions
• T. harzianum likes warm conditions
• Tolerant of low moisture
• Tolerant to many fungicides
• Prefers acidic soils (pH 3.5 to 4.5)
• Some are not rhizosphere competent

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Trichoderma spp.

• Mode of action
• Diffusible inhibitors (toxins, antibiotics)
• Volatile inhibitors (alcohols, ketones,
  sesquiterpenes)
• Competition
• Mycoparasitism
• Chemotaxis to target
• Galactose residues (T) bind to lectins on host
• Coiling
• Extracellular enzymes (cellulases and chitinases)
• Appresoria like structures and penetration

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From - http//www.nysaes.cornell.edu/ent/biocontro
l/pathogens/trichoderma.html
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Trichoderma Tricks

• Problem Rhizosphere incompetence
• Genetic improvement
• Mutation Papavizas et al., 1982 Papavizas and
  Lewis, 1983
• UV irradiation, 80 minutes, V-8 juice agar
• Benomyl, thiabendazole, thiphanate-methyl
  tolerant
• Rhizosphere competent
• Target specificity
• R1 and R2 - Fusarium oxysporum f. sp. melonis
• R3 and R6 - superior Pythium ultimum parasite
• R5 and R6 - superior Sclerotium rolfsii
• Protoplast fusion between strains

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Sporidesmium sclerotivorum

• In culture, this Deuteromycete, produces
• macroconidia, microconidia, chlamydospores,
  microsclerotia
• In nature, behaves as an obligate parasite
• Parasitizes sclerotia of Sclerotinia
  sclerotiorum, Sclerotinia minor, Sclerotinia
  trifoliorum, Sclerotium cepivorum, Botrytis
  cinerea
• Originally isolated in Beltsville, MD
• 22 kg/ha

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Lettuce Drop

• Pathogens
• Sclerotinia minor, S. sclerotiorum

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http//www.ncsrp.com/whitemold/bioconwm.htm
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http//www.ncsrp.com/whitemold/bioconwm.htm
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Sporidesmium sclerotivorum

• How it works
• Macroconidia germination when nearby sclerotia
• Germ tube infection, sclerotial matrix
  penetration
• S. sclerotivorum stimulates sclerotia glucanase
  activity (food)
• Feeding haustoria
• 15,000 macroconidia
• Needs moisture, 20-25 C, pH 5.5-7.5

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Bacteria

• Types
• Soil saprophytes
• Bacillus spp.
• Fluorescent pseudomonads
• Plant pathogenic
• Parasites
• Facultative parasites

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Principles

• Selection of candidate bacteria
• Schisler, D. A. and Slininger, P. J. 1994.
  Selection and performance of bacterial strains
  for biologically controlling Fusarium dry rot of
  potatoes incited by Gibberella pulicaris. Plant
  Dis. 78251-255.
• Formulation
• Mechanisms of pathogen suppression
• substrate competition and niche exclusion
• siderophores
• antibiotics
• induced resistance

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Bacterial Products
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Bacterial Products
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Bacterial Products
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Bacterial Products
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Bacterial Agents for Fungal Disease Management

• Biological control studies with bacteria has
  examined for over 70 years
• Sources of biological control bacteria
• Suppressive soils
• On aerial plant parts (epiphytes, phylloplane)
• On root surfaces (epiphytes, rhizoplane)
• Colonizing plant pathogens (hyperparasites)
• Plant disease causing bacteria (phytopathogens)

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Some Examples

• Bacillus subtilis, Kodiak Quantum 4000,
  Gustafson, first Plant Growth Promoting
  Rhizobacterium (PGPR)
• Streptomyces griseoviridis, Mycostop
• Pseudomonas syringae Bio-save100
• Pseudomonas fluorescens, P. fluorescens 2-79
• Burkholderia cepacia, Deny, Intercept, Blue
  Circle
• Pseudomonas corrugata (my work)

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Bacillus

• Bacillus species are soil bacteria that
• Produce endospores
• Produce a wide range of antibiotics (70)
• Species known to produce antibiotics
  licheniformis, pumilus, circulans, cereus,
  Brevibacillus laterosporus, Paenibacillus
  polymyxa
• Shown to have antibacterial and antifungal
  activity against phytopathogens
• Swinburne, et al. 1975 antibiotic activity,
  suggested alternative or supplementary use to
  chemical pesticides
• Katz and Demain, 1977 peptide antibiotics
• Shoji, 1978 peptide antibiotics
• Produce compounds that stimulate plant defenses
• Phenylalanine ammonia-lyase (PAL)

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Bacillus subtilis Epic, Kodiak, Rhizo Plus,
Serenade, System 3

• Bacillus subtilis A13
• Registered on peanut in 1988
• Registered on cotton and broad bean in 1990
• Background
• Broadbent et al., 1977
• Inhibited fungi (Phytophthora spp., Pythium spp.,
  Fusarium spp., Sclerotium spp., Rhizoctonia spp.)
• Stimulated growth of eggplant, dahlia and cabbage
  in steamed soil
• Seed treatment Carrots (48), Oats (33),
  Peanuts (37) yield increases

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Kodiak

• Biological Control Agent Bacillus subtilis
• Target Pathogen/Disease Rhizoctonia solani,
  Fusarium spp., Alternaria spp., and Aspergillus
  spp. that attack roots
• Crop cotton, legumes
• Formulation dry powder usually applied with
  chemical fungicides
• Application added to a slurry mix for seed
  treatment hopper box treatment

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Streptomyces
Streptomyces, is an actinomycete, non-motile,
filamentous, Gram-positive, spore forming (borne
on sporophores aerial spore bearing filaments).
They are found worldwide in soil where they
contribute much of the earthy smell by
production of geosmens. Streptomyces is
metabolically diverse and have uses for
antibiotic production, bioremediation, and in
biological control. A few species (out of over
500) are pathogenic in plants and animals.
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Pseudomonads

• Pseudomonads are Gram negative, aerobic,
  heterotrophic, bacteria that are motile by polar
  flagella
• Some produce fluorescent pigments
• These are placed in a separate subgroup of the
  genus Pseudomonas (species fluorescens,
  aeruginosa, syringae, putida
• Some are opportunistic human pathogens (P.
  aeruginosa)
• Epiphytes and pathogens of plants (P. syringae)

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Pseudomonas syringae

• Genus and species that contain over 41 pathovars
  that cause disease on various plants
• Produce toxins, proteases, cellulases, lipases,
  etc.
• Fortunately, there are strains of P. syringae
  that are antagonistic to pathogens

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Pseudomonas syringae

• Janisiewics, W. 1987 isolated P. syringae
  antagonist strain from apple leaves. Also found
  on apple, pear, and citrus fruits.
• The bacterium along with fungal yeasts, are
  efficient colonizers of wounds without being
  pathogenic.
• Can prevent infection by fungal pathogens
  (Botrytis, Penicillium, Fusarium).
• Can prevent infection by foodborne bacterial
  contaminants (E. coli 0157LH7).

http//www.nysaes.cornell.edu/ent/biocontrol/patho
gens/pseudomonas_s.html
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Pseudomonas fluorescens

• Several products use strains of P. fluorescens
• BlightBan A506
• Was originally developed for fire blight, a
  bacterial disease of stone fruit trees.
• Conquer, Victus
• For control of P. tolassii (brown blotch) in
  mushrooms
• MVP II and Mattch mixtures of P. fluorescens
  and Bacillus thuringiensis
• Protectant for the endotoxins
• Frost Ban

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Pseudomonas fluorescens

• Weller and Thomashow
• 2,4-diacetylphloroglucinol (PHL)
• Produced by many fluorescent Pseudomonas spp.
• Groups of strains
• Produce PHL, pyoluteorin, and HCN
• Produce PHL, HCN
• Phenazine-1-carboxylic acid
• Strains 2-79 and 30-84 (P. aureofaciens).
• Antibiotic is produced on roots
• More antibiotic is produced on roots grown in
  steam sterilize soil
• Roots with antibiotic production vs. non treated
  and antibiotic mutants had greater disease
  suppression (take-all disease Gaeumannomyces
  graminis var. tritici)

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Burkholderia cepacia

• Formerly known as Pseudomonas
• 1949 described by Walter Burkholder as a pathogen
  of onions.

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Burkholderia cepacia

• Nutritionally versatile, Gram-negative, pathogen
  of onions
• Found in soil and in moist environments
• Opportunistic human pathogen in hospitalized,
  immunocompromised, and cystic fibrosis patients
• Can degrade chlorinated aromatic substrates (ex.
  2,4,5 chlorophenoxy acetic acid)

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Burkholderia cepacia

• Antagonist of many soil borne plant pathogens
• Alternaria, Aphanomyces, Botrytis,
  Cylindrocarpon, Fusarium, Pythium, Rhizoctonia
• Antifungal compounds
• Cepacin A and B (Parker et al. 1984. J. Antibiot.
  37431-440)
• Cepalycin (Abe and Nakazawa. 1994. Microbiol.
  Immunol. 381-9)
• Pyrollnitrin (Homma et al. 1989. Soil Biol.
  Biochem. 21723-728)

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Burkholderia Products

• Blue Circle, Deny, Precept,
• Soil Pathogens Fusarium, Pythium
• Nematodes lesion, spiral, lance, and sting
• Crops alfalfa, barley, beans, clover, cotton,
  peas, grain sorghum, vegetable crops, and wheat.
• Genetically versatile, has 4 replicons allowing
  for homologous and illegitimate recombination.

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Discovering a New Agent
In California, 90 of the production of fresh
market tomatoes occur in 9 counties, of these,
San Diego County is the forth largest with about
4.25 thousand acres. Much of the fresh market
production involves stake (pole) tomatoes. For
some producers virgin land is leased and used for
production for 3 years.
Pruning is a management technique used to promote
early and continuous fruiting while training the
vine for an upright growth habit.
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Pathogen Issues
http//ohioline.osu.edu/hyg-fact/3000/3122.html
In virgin land and in crop rotated (continuous
vegetable production land), Fusarium wilt
(Fusarium oxysporum f. sp. lycopersici) is a
sporadic problem.
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Pathogen Issues
The other problem was bacterial canker caused by
Clavibacter michiganensis subsp.
michiganensis. However, in 1981 a new disease was
discovered in the field.
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Pseudomonas corrugata

• Non-fluorescent, opportunistic pathogen
• Tomato pith necrosis
• By 1982, the disease had become the number one
  concern of the fresh market tomato producers in
  San Diego county and begun to appear in other
  counties as well

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Table 1. Phytopathogenic fungi that are
inhibited in vitro by Pseudomonas corrugata.
a slight, moderate, strong.
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Table 2. Antagonism of bacterial plant
pathogens by Pseudomonas corrugata in vitro. Two
day old spot cultures of P. corrugata were
chloroform killed and overlaid with top agar
containing the test bacteria.
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Summary

• Mycoparasites
• Obligate parasites
• Selective to broad range
• Bacteria
• Broad range
• Saprophytes
• Facultative parasites