Plant pathogens and microbial pest control

Question 1

What do biological control agents (BCAs) do?

Answer 1

Suppress pathogen by affecting its growth and spread directly by parasitism.
Occupy niches to prevent pathogens from colonising the plants (indirect).
Induce plant defence responses (host-mediated) - increased plant immunity

Question 2

Describe how Ampelomyces quisqualis can be used as BCAs.

Answer 2

Produce pycnidia atop mildew hyphae and conidiphores, eventually destroying mildew colony - penetrate hyphae of powdery mildew fungi growing internally from cell to cell through the septal pores.
Spores are formulated into water-dispersible granules that are sprayed onto grapes when they germinate and colonise the grape powdery mildew fungus to kill it

Question 3

Describe how Trichoderma spp. can be used as BCAs.

Answer 3

Can protect plants against soil-borne fungal pathogens by occupying niches around plant roots and/or adhering to roots and hyphae form protective layer.
Can rapidly absorb nutrients required for growth of pathogenic fungi, resulting in nutrient deficiency and inhibiting growth and reproduction.
Direct: Invade mycelium causing breakdown of pathogen cell wall. Feed on fungal host, producing hydrolytic enzymes and antimicrobial peptides to degrade cell wall and make holes in lipid membrane so host fungal cells lyse. Accumulated peptobiols embed vertically in the cell membrane.

Question 4

Describe how Phebiopsis gigantea can be used as a BCA.

Answer 4

Fungus Heterobasidian annosum enters plantations through fresh stump tops and spreads across root contacts into live trees which die/rot.
Chemicals for treatment are limited.
P. gigantea if saprophytic fungus which causes white rot of conifer stumps. Doesn’t attack living trees but when applied to stumps, colonises it and prevents surrounding trees from becoming infect with H. annosum by competing for resources on stump surface - hyphal interference, antagonise other hyphae

Question 5

Describe how hypovirulent mycoviruses can be used as a BCA to treat chestnut blight.

Answer 5

Fungus Cryphonectria parasitica grows in inner bark and cambium.
Hypovirulent strains give small necrosis but are unable to overcome tree’s defences and is restricted to superficial area of the bark - due to mycovirus.
Mycovirus can be transferred to aggressive isolates via sexual reproduction (anastomosis - fungal cells fusing together, mixing of cytoplasm but requires compatibility of mating types).

Question 6

Describe how Bacillus subtilis can be used as a BCA.

Answer 6

Colonises developing root system of plant and competes with certain fungal disease organisms. Also produces enzyme AiiA (inactivates quorom sensing in pathogen Pectobacteria) - gene engineered into potato plants where confers high level of resistance against Pectobacteria.
Spores incorporated into biocontrol products against a range of fungal pathogens.

Question 7

Describe how bacillus thuringiensis (bt) can be used as a BCA.

Answer 7

Spore-forming bacterium that makes insect toxins. Marketed for control of pests such as caterpillars, butterflies, moths and beetles.

Question 8

Describe pseudomonas fluorescens as a BCA.

Answer 8

Produces antimicrobial secondary metabolites which inhibit plant pathogens - mutant with inability to produce antimicrobials are ineffective. Incorporated into products for control of damping-off diseases

Question 9

Describe Argobacterium radiobacter as a BCA.

Answer 9

Soil-inhabiting bacterium that doesn’t infect plants. Some strains produce an antimicrobial compound (agrocin) which kills certain pathogenic strains of A. tumefaciens (causes crown-gall disease - unregulated growth of plant cells, tumour, plant dies, bacteria released in soil, new infection cycle starts)

Question 10

What is the difference between system acquired resistance (SAR) and induced systemic resistance (ISR)?

Answer 10

SAR = responses generated through plant cell necrosis
ISR = resistance induced by plant growth promoting rhizobacteria and mycorrhizal fungi
Both mechanisms result in signals that induce gene expression and resistance in distal parts of plants.

Question 11

What are baculoviruses?

Answer 11

Large DNA viruses that infect insects; ubiquitous in environment; control insect populations naturally.

Question 12

Describe Occlusion-derived virions (ODV).

Answer 12

Occur when mature virions are embedded in a proteinaceous crystal matrix consisting of mainly one single viral protein polyhedrin.
ODV within Occlusion body (OB) spread virus between insects - horizontal transmission - stay in nucleus until cell lyses.
ODBs (Occlusion-derived bodies) stay in nucleus where packed into OB’s

Question 13

Describe Budded Virions (BV).

Answer 13

Occur when single nucleocapsid exists nucleus and buds from cell acquiring an envelope from the plasma membrane. Spread infection from cell to cell.`

Question 14

Describe the life cycle of baculoviruses.

Answer 14

Insect eats infected plant, OB dissolve in highly alkaline environment of insect midgut and ODVs are released.
ODVs attach to microvilli of midgut epithelial cells.
Infected midgut epithelial cells produce BVs that use tracheal cells to escape midgut and infect rest of insect

Question 15

Describe secondary infection of baculoviruses (from cell to cell).

Answer 15

BVs enter cell through endocytosis.
Nucleocapsid is transported to the nucleus.
DNA inside nucleocapsid is released (uncoating) and replicated in the nucleus (DNA replication, translation and nucleocapsid assembly happen in nucleus).
Nucleocapsid leaves nucleus, acquiring the envelope from the nuclear membrane that is eventually lost.
Nucleocapsid travels to plasma membrane where bud off acquiring envelope, forming BVs

Question 16

Describe how baculoviruses are classified by OB morphology.

Answer 16

Single-nucleopolyhedrovirus (SNPV) - 1 single capsid within an envelope and embedded in an OB made of polyhedrin.
Multi-nucleopolyhedrovirus (MNPV) - several capsids within an envelope and embedded in an OB made of polyhedrin
Granulovirus (GV) - 1 capsid within an envelope and embedded in an oval OB made of granulin rather than polyhedrin

Question 17

Describe the effect of BV on larvae molting.

Answer 17

EGT (ecdysteroid UPD-glucosyltransferase) inactivates ecdysone (major insect molting hormone). Molting is a very stressful process and pathogen wants to keep animal feeding and growing so it can infect more cells and then more animals.

Question 18

Describe wandering behaviour in larvae as a result of BV infection.

Answer 18

ptp (protein tyrosine phosphatase) gene responsible for enhanced locomotion activity.
Virus manipulates brain of caterpillar to make them wander and climb - when larvae die and liquify, the OBs can be dispersed more easily

Question 19

What are the limitations of using BVs for pest control?

Answer 19

Low speed of kill - advantageous for virus but larvae keep eating.
Production of BVs is labour intensive - need a living host, specific species sometimes
Sensitive to UV light

Question 20

Describe endospore formation in Bacillus thuringiensis (Bt).

Answer 20

Replication of bacterial chromosomes, cells divide asymmetrically.
Large portion of cell is mother cell in charge of nourishing developing spore (forespore).
Mother cell engulfs forespore completely so it is surrounded by two cell membranes.
Layer of murein (cortex) synthesised between the two membranes.
Forespsore becomes surrounded by protective layers and synthesises dipicolinic acid.
Mother cell lyses, releasing mature endospore.

Question 21

What proteins are produced by Bt and in what stages?

Answer 21

Cry (crystal) and Cyt (cytosolic) are produced during sporulation and stationary phase.
Vip (vegetative insecticide protein) and Sip (secreted insect toxic protein) produced during vegetative growth phase.

Question 22

How are Cry proteins toxic to insects but safe for humans contact?

Answer 22

Make pore in the caterpillar gut but part of the protein has to be cleaved to become toxic. Cleavage only happens in alkaline conditions

Question 23

Describe what is known about Cyt toxins.

Answer 23

Two mechanisms of action suggested: pore-forming and detergent action mechanism (make membrane more permeable).
Cause organisation of gap epithelium and insect death.
Classified into 3 families: Cyt 1-3

Question 24

Describe how the four families of Vip are toxic to insects.

Answer 24

Vip1/2: Translocated into cytoplasm by acid endosomes - destroys actin filaments and cell dies due to cytoskeletal disarrangement (against Coleoptera and Hemiptera)
Vip3: Binds midgut epithelial cells (against Lepidoptera)
Vip4: Toxicity unknown and species affected unknown

Question 25

Describe the current evidence for Bt crops being safe.

Answer 25

The bacterium Bt occurs naturally in the soil.
Bt spore-based products contain Bt toxins too - fine to use in biological agriculture.
Toxins are protoxins and require proteolytic cleavage in alkaline insect midgut to turn into active toxin.
Toxin needs to be able to bind to specific insect receptors to form pores or induce cell death.
Food is processed and heat inactivates Cry toxins.
Most ingested dietary proteins, including Cry, undergo degradation in GI tract

Question 26

Describe how the fungus Strongwellsea spp. spreads infection through living ants

Answer 26

Consumes genitals, fat reserves, reproductive organs and finally muscles while host continues to live normally for days. The whole time, it shoots thousands of spores through holes in abdomen onto other individuals

Question 27

Describe how Massospora cicadina infects and spreads disease through periodical cicadas.

Answer 27

Infection results in plug of spores that replaces end of abdomen while still alive, leading to infertility, disease transmission and eventual death of cicada.
Increases sex drive of male cicadas.
Also makes infected males flap their wing like females do and other males try to mate with them. Infection spreads through copulation attempts

Question 28

How do entomopathogenic fungi infect insects by contact?

Answer 28

Spore germinates on top of cuticle
When insect is dead fungus grows outwards and sporulates in the cadaver before infecting other insects

Question 29

What are the benefits of using entomopathogenic fungi compared to chemical pesticides?

Answer 29

More environmentally friendly
Lower health risks
More sustainable
Lower non-target effects
Greater specificity

Question 30

What are the obstacles to using entomopathogenic fungi as biopesticides?

Answer 30

Cost
Reliability
Shelf life (dealing with living organisms)
Environmental factors (variable efficacy results)

Question 31

How can entomopathogenic fungi be used against fruit flies?

Answer 31

Eggs are laid under the skin of the fruit and larvae stay inside of the fruit and cannot be targeted with microbial pest control.
When they are ready to pupate, they jump off the fruit and pupate in the soil next to the tree.
Can apply fungi to the soil to target the soil dwelling stage and prevent adult emergence.

Question 32

What is the difference between a pheromone and a parapheromone?

Answer 32

Pheromone = volatile chemical substance produced and released into environment by an animal which affects the behaviour of others within the same species.
Parapheromone = volatile chemical (or blend) that mimics the effect of a pheromone

Question 33

Describe Male Annihilation Technique using an example.

Answer 33

MAT - using male-specific attractants to get rid of males, preventing from mating with females.
Used to suppress and eradicate populations of fruit flies. Male flies trapped using lure baits or bait stations containing male attractant and insecticide (can be substituted by a mycopesticide - more environmentally friendly)