Nematodes and Plant Damage

Rev 10/30/2019

Function of roots

• absorb water
• absorb nutrient ions
• storage
• anchorage
• dynamic mining function, regeneration.

Terrestrial plants are estimated to have annual production of 120 billion tons of biomass - 5% minerals = 6 billion tons of minerals mined from the soil each year.
 

A plant may transpire its own weight in water in a day.
 

The integrity of Casparian strip (waxy layer around endodermis cells) is important - nutrients, and sometimes water, are taken up against a gradient.

Secondary roots and endoparasites are disruptive to the Casparian strip.

Components of Damage

A. Demand

Total energy consumption during the lifecycle of a female root-knot nematode (Meloidogyne spp.) is 1 calorie.

The total biomass of a female root-knot nematode is 200 µg, including the egg mass (Melakerberhan and Ferris).  For say 100,000 nematodes in a root system, the total nematode biomass is 20 g! Allowing for 50% production efficiency, total material extracted from the plant would be 40 g. So, the demand effect on the plant may be minimal unless plant is very stressed and resources are limited.

An adult Heterodera schachtii consumes 11 nL/day of cell content (Muller et al, 1981).  So, it would take 1,000,000 such females to remove 11 ml of cell content in a day.

B. Mechanical Disturbance

a. Penetration of cells - relative to length of stylet. Damage will depend on types of cells affected - storage tissues, cortex, or functional vascular.

b. Migration through tissues - intercellular and intracellular requiring dissolution of cell walls, middle lamellae. Suggests cellulase and pectinase enzymes - spongy tissues, sloughing, e.g. damage caused by Pratylenchus and Ditylenchus.  Allows ingress of other organisms.  Root-knot (Meloidogyne spp.) and cyst (Heterodera spp.) produce endogluconase (cellulase) enzymes and pectate lyase which are presumable involved in the passage through plant tissues.

c. Leakage from damaged tissues - it is estimated that up to 20% or more of photosynthate partitioned to roots may leak into rhizosphere soil without root damage. "Root exudation" nurturing organisms in rhizosphere - presumably to plant benefit - but speculate that selection has optimized the costs and benefits. Enhancing root leakage through nematode damage must reduce plant productivity.

C. Physiological Disturbance

a. Nematode secretions - associated with establishment and maintenance of feeding sites. Effects increase with sedentary endoparasitism.  Secretions from the nematode digestive glands may polymerize into a feeding tube inside the cell.  The feeding tube remains associated with the stylet during ingestion.  When the stylet is withdrawn the opening in the cell wall is sealed with an electron-dense feeding plug.

• Cell wall permeability and surface enlargement - transfer cells, cell wall thickening.
• Increase in cytoplasm density, metabolic activity.
• Hypertrophy - cell enlargement, 500 to 1000x increase in volume (Meloidogyne) - nuclear division without cytoplasmic division (karyokinesis without cytokinesis). But different strategies in different genera - eg cell wall dissolution in Heterodera. Multinucleate cells, nuclei larger.
• Hyperplasia - tissue enlargement - mitotic activity -galling, root tip galls etc.
• Disruption of normal meristem activity - Xiphinema, Trichodorus, Hemicycliophora (different taxonomic groups). Damage to meristems.
• Physiological control of carbohydrate partitioning - Metabolic sink - McClure, ¹⁴CO₂ studies.

b. Physiological effects -  

• increased root respiration (more mass), 
• increased growth respiration - repair, 
• mobilization of defense mechanisms.
• increased levels of plant hormones (indole acetic acid, cytokinins) in galled tissue, but source uncertain.

c. Whole-plant effects - Disturbance of the biochemical network. Wallace (1987) points to the complexity of the biochemical pathways:
Photosynthesis divided into two basic phases - a light phase when light energy is converted into chemical energy, and a synthetic phase in which carbohydrates are formed in a series of reactions accelerated by light. Photosynthesis involves a chain of metabolic events cross-linked to other physiological processes, so disruption of one may have effects throughout system.
For example, Bird suggested that photosynthesis is reduced in tomato by Meloidogyne javanica by inhibiting production of cytokinins and gibberellins in roots, and/or by increased stomatal resistance due to water stress.
Fatemy et al. indicate that the response of potato to Globodera rostochiensis is due to stomatal closure through water stress; the result is reduced photosynthesis.
However, generally the mechanisms by which root-infecting pathogens, including nematodes, affect physiological processes have been insufficiently studied.

d. Plant as an Integrator - Metabolic pool concept - plant as an integrator - concepts of demand and damage. Melakeberhan and Ferris characterized five effects of root-knot nematode infection in grape while exploring the impact in an energy partitioning and flow model:

• Reduction (disruption) of water uptake. Seinhorst, however, asserted that there is little evidence of reduction of water uptake in response to nematodes. He measured daily water uptake on a water usage basis - rate of water loss from pots minus increase in dry wt minus evaporation from surface. The rate was a linear function of total dry wt. So, he argued that rate of use per g tissue is constant. However, root damage could result in lower water uptake, and final dry weight could be a function of the rate of water uptake.
• Reduction in rate of photosynthesis.
• Reduced leaf expansion and total photosynthesis.
• Alteration of partitioning of photosynthate- change in root/shoot ratio.
• Increased leakage - direct effect and affect on other pathogens energy supply (Garrett - inoculum potential as a function of the abundance of infective units and the energy resources available to them).

D.  Molecular Events:

Successful Parasitism by Plant-parasitic Nematodes

Suppression of Plant Defenses by Nematodes

(Powepoint overview) 

Biotrophic Pathogens

• Pathogens that can only feed on a living host and must keep the host and its cells alive are termed biotrophic pathogens.

• Sedentary ecto- and endoparasitic nematodes are in this category, for example, species of Meloidogyne, Heterodera, Xiphinema, Tylenchulus, Rotylenchulus.

• Nematodes that withdraw contents form individual cells and then move to new feeding sites are considered cell grazers.

 Innate Immunity

• Most plants are resistant to most pathogens; they have highly effective immune systems.  Host defense mechanisms may be as extreme as programmed cell death, the hypersensitive response.

• All biotrophic pathogens must suppress host defenses.  The feeding site must be induced without host detection or without induction of host defenses. 

• Following establishment of the feeding site by sedentary nematodes, it must be maintained for up to 5 or 6 weeks to allow the nematode to achieve its reproductive potential. 

• That time scale is much greater than that required by many bacterial and fungal pathogens of plants. 

• Failure to establish and maintain the feeding site may prevent reproduction and therefore is catastrophic to the nematode genotype.

• Consequently, there is strong selection pressure on nematodes to suppress host defenses.

Plant Defenses

1. Pre-existing Defenses – Basal Resistance

a.    Structural – cuticle, wax, wall thickness, spines that suppress penetration of cells.

b.    Chemical-phenolic and other compounds that inhibit or kill invading organisms.

    2.  Systemic Induced Resistance

1. PAMP Signals

• Organisms attempting to feed on plant cells, or to invade plant tissues, betray their presence with recognizable molecular signals on their surfaces. 

• Such signals are termed pathogen associated molecular patterns (PAMPs) and are recognized by pattern recognition receptors on cell surfaces. 

• Chitins in fungal cell walls are PAMPS which trigger immunity responses (pathogen-triggered immunity – PTI). 

• The PAMP signals of nematodes are unknown; chitin is not present in the cuticle although it does occur in egg shells and perhaps in the stylet. 

• Plants characteristically deposit callose to strengthen cell walls at the point of invasion, including at the point of nematode stylet insertion.

• Also, pathogen invasion trigger the triggers jasmonic acid signaling pathway which stimulates production and release of other defense toxins.

    b. DAMP Signals

Another set of signals that may trigger PTI responses in plants are cell-degradation products resulting from damage caused by the invasion, damage-associated molecular patterns (DAMPs).

 Effector Suppression of Plant Defenses and PTI

Invading bacteria and fungi, and probably nematodes, release effector molecules into plant cells to suppress PTI and render the plant susceptible to infection or invasion. 

PAMP-triggered PTI, the first line of defense, may involve production of salicylic acid (SA) as a signal to invoke defense mechanisms.  In that case, successful nematode infections would involve suppression of SA production, reduction of callose thickening of cell walls and suppression of active oxygen defense responses (H₂O₂, superoxide) which may initiate localized programmed cell death – hypersensitive response.

SA signaling is possibly disrupted by chorismate mutase produced in the esophageal glands.  In the PTI signaling pathway, chorismate is converted to salicylic acid.  Chorismate mutase from the nematode reduces chorismate and thus SA, so defense mechanisms are not triggered.  Incidentally, like cellulases, chorismate mutase is an example of horizontal gene transfer from bacteria. Nematodes are the only metazoan with the enzyme.

An alternative mechanism of PTI suppression by nematodes is the production of effectors which cause ubiquitin to attach to plant signal proteins and thus reduce their levels and effectiveness in triggering PTI responses.

The evolution of effector suppression of PTI has resulted in evolution of immune receptors, with a nucleotide-binding domain and a leucine-rich domain (NB-LRR), in plants that recognize the effector molecules and activate effector-triggered immunity (ETI).  However, successful pathogens have evolved next-generation effectors that suppress ETI. 

One possible candidate is the Hg30C02 effector protein of Heterodera glycines which may be involved in active suppression of host defenses (Hamamouch et al., 2012).  

Another is the 8D05 parasitism gene of Meloidogyne incognita which is required for successful infection of host roots.  The gene codes for a protein that is secreted from the subventral glands duriing initiation of the feeding site (Xue et al., 2013).

Plants have responded with more specific ETIs and the evolutionary treadmill continues. 

PTI responses to PAMPs and DAMPs are relatively general in their effect but higher level ETIs are progressively more specific to individual pathogens. 

The cyclical evolutionary process of plant-nematode interactions with regard to plant immunity and susceptibility is depicted by the zig-zag-zig model (Jones and Dangl, 2006).  Initially PAMPs trigger PTI which reduces susceptibility.  Then nematodes develop effectors that suppress PTI and plants evolve immunity responses to the effectors.

The Evolutionary Response: Effector-triggered Immunity (ETI)

In effect, the sources of specific ETIs are resistance genes.  Thus, the Mi gene of tomato codes for receptors to the effector molecules introduced by root-knot nematodes to suppress plant defenses and, perhaps, facilitate the development of feeding sites. 

However, although they are known for some other pathogens, the nematode effector molecules that trigger ETIs have not yet been determined and are the focus of several active research programs.

Although the nature of nematode effector products has still to be determined, the hypersensitive response of cells to the activated ETI effectively disrupts the feeding and development of sedentary endoparasitic nematode species.

Suppression and Avoidance of Host Defenses

 Nematodes are protected by the cuticle and surface coat.  The surface coat of lipid and protein molecules is shed as the nematode moves, shedding bacteria but also confusing the plants as to its whereabouts.

Glutathione peroxidases on surface coats reduce active oxygen plant defenses.

Many plant-parasitic nematodes produce glutathione S tranferases that detoxify endogenous toxic molecules.  They also produce superoxide dismutase that breaks down active oxygen plant defenses.

A recent (2011) compilation of the understanding of actions of Nematode Effector Proteins in host plant cells

The above compilation by Gheysen and Mitchum (2011) is based mainly on research on cyst and root-knot nematodes.   Cell wall degrading enzymes (CWDEs) and chorismate mutase are secreted by both root-knot and cyst nematodes while 16D10 is specific for root-knot nematodes. Effector proteins originating from the subventral and dorsal esophageal gland cells are secreted into plant tissues through tthe nematode stylet. CWDEs facilitate nematode migration through root tissues. Effector proteins containing a nuclear localization signal affect the plant nucleus. Nematode secreted ubiquitin extension proteins may alter cellular protein degradation pathways or function as signaling molecules. Chorismate mutase changes the subcellular balance of chorismate. Upregulation of the auxin influx transport proteins, AUX1 and LAX3, and downregulation of PIN1 leads to a local accumulation of auxin (IAA) in the initial feeding cell. Relocalization of PIN3 to the lateral plasma membranes delivers auxin to adjacent cells. LAX3 regulates auxin influx in adjacent cells stimulating cell wall hydrolysis for subsequent incorporation into the developing feeding site. Other putative nematode effectors  include proteases, venom-allergen proteins (VAPs),calreticulin, MAP-1, RBP-1, and NodL. Synopsis from Gheysen and Mitchum (2011).

E. Evolutionary Events:

The genome of plant-feeding nematodes of the sub-order Tylenchina includes genes that encode for endoglucanases. Endogluconases are cellulases, a family of enzymes formerly thought to be restricted to prokaryotes. Other plant-cell wall digesters such as termites and ruminants use symbiotic and commensal bacteria to achieve dissolve cellulose.  The presence of these and other genes suggests that horizontal or lateral gene transfer has occurred between bacteria and nematodes.

F.  Proof of pathogenicity:

Koch, Pasteur - the germ theory - required rules of proof.

Mountain provided guidelines for obligate parasite nematodes:

• - establish association - ecological phase
• - establish parasitic capabilities - biological phase
• - establish host-parasite relations - etiological phase

Predisposition

Interaction with other organisms

• a. other nematodes
• b. fungi
• c. bacteria
• d. viruses
• e. abiotic/physical stresses

Note - term "interaction" is loosely used - implies that effect in combination is different than sum of individual effects - not additivity. Three descriptions of the result of combinations of organisms: -synergistic, -suppressive, -no interaction.

Mechanisms of interactions

        Synergistic:

• Reduced tolerance - multiple stress
• Vectoring - Longidoridae and Trichodoridae
• Change in substrate - fungi?
• Routes of ingress - fungi, bacteria
• Leakage - energy source - fungi, bacteria

        Suppressive:

• Induced resistance - any examples?
• Reduction of stress - mycorrhizae
• Biological antagonists - but not an interaction of two pathogens
• Reduced substrate availability

Importance of Nematodes in World Agriculture

See Sasser and Freckman in Vistas on Nematology.
Questionnaires returned by 371 nematologists worldwide (handout with Tables 2 and 3)
Consider inherent biases in data of this kind.
Consider means and variance in crop loss data.
- almost nobody had 10% yield loss
- include management costs as part of the loss

Ranking of important genera and relative weight:

Note - considerable variation by region, so questionnaire data biased by number of respondents per region.

International Survey of Crop Losses due to Nematodes

 Crop losses - measurement and estimates

• - Problem of reference yield - how do you isolate one pest effect?
• - Need for holistic approach.
• - How to remove one stress from system without changing system.

Estimated Crop Losses 2008

Crop	Number of estimates per crop	FAO production estimates (1000 metric tons)	Estimated price per metric ton -2008 (U.S.$)	Estimated yield losses due to nematodes (%)	Estimated monetary loss due to nematodes - 2008 (x1000 U.S.$)
Banana	78	81,263	928	19.7	14,855,056
Barley	49	136,209	238	6.3	2,044,978
Cassava	25	228,138	175	8.4	3,353,629
Citrus	102	105,000	711	14.2	10,601,170
Cocoa	13	4,012	2,693	10.5	1,134,626
Coffee	36	7,742	1,915	15	2,223,425
Corn	125	637,444	183	10.2	11,895,929
Cotton (lint only)	85	112	1,040	10.7	12,463
Field bean	70	6,371	1,200	10.9	833,327
Oat	37	25,991	117	4.2	127,327
Peanut	69	30,670	1,470	12	5,410,188
Potato	141	321,736	264	12.2	10,362,473
Rice	64	432	624	10	26,957
Sorghum	53	64,589	59	6.9	262,942
Soybean	91	56,389	339	10.6	2,024,967
Sugar beet	51	247,878	47	10.9	1,258,234
Sugar cane	65	1,557,664	36	15.3	8,462,835
Sweet potato	67	126,299	407	10.2	5,242,210
Tea	16	3,871	282	8.2	89,637
Tobacco	92	6,326	6,600	14.7	6,137,485
Wheat	89	676,300	237	7	11,237,807
Sources:
http://www.fas.usda.gov/wap/circular/2008/08-09/productionfull09-08.pdf
http://usda.mannlib.cornell.edu/usda/current/PeanPrice/PeanPrice-10-10-2008.pdf
http://usda.mannlib.cornell.edu/usda/current/CropProdSu/CropProdSu-01-11-2008.pdf
http://www.nass.usda.gov/Publications/Ag_Statistics/2008/index.asp

References:

Bird, A.F.

Davis. E.L., R.S. Hussey, T.J. Baum, J. Bakker, A. Schotts, M.N. Rosso and P. Abad. 2000. Nematode parasitism genes.  Ann. Rev. Phytopathol. 38:365-396.

Dropkin, V.

Garrett

Gheysen, G. and Mitchum, M.G. 2011. How nematodes manipulate plant development pathways for Infection. Current Opinion in Plant Biology 14:415-421.

Hussey, R.S. and V.M. Williamson 1998.  Physiological and molecular aspects of nematode parasitism.  Agronomy Monograph 36.

Koenning, S.R.,  Overstreet, C., Noling,, J.W., Donald, P.A., Becker, J.O., Fortnum B.A. 1999. Survey of Crop Losses in Response to Phytoparasitic Nematodes in the United States for 1994.  Journal of Nematology 31:587-618.

Melakeberhan and Ferris

McClure

Muller et al, 1981

Sasser, J.N., Freckman, D.W., 1987. A world perspective on Nematology: the role of the society.  Pp 7-14 in J.A. Veech and D.W. Dickson (eds) Vistas on Nematology. Society of Nematologists, Hyattsville, Maryland. 509p.

Seinhorst

Wallace, 1986

Wyss U., F.M.W. Grundler and A. Munch. 1992. The parasitic behaviour of second-stage juveniles of Meloidogyne incognita in roots of Arabadopsis thaliana. Nematologica 38:98-111.

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