Management of Soil-Borne Fungi and Root-Knot Nematodes in Cucurbits through Breeding for Resistance and Grafting

Agronomy Review: Management of Soil-Borne Fungi and Root-Knot Nematodes in Cucurbits

Abstract

Soil-borne pathogenic fungi (SBPF) and root-knot nematodes (RKN) are major pathogens in cucurbits, co-existing in the rhizosphere.
Plant resistance is effective for controlling soil-borne diseases; proper pathogen diagnosis is crucial for selecting resistant cultivars or rootstocks.
Plants defend themselves against SBPF and RKN via cell wall hardening, pathogenesis-related (PR) proteins, and antimicrobial molecules.
Plant hormones like salicylic acid, jasmonic acid, and ethylene play a role in cucurbit responses to SBPF.
Most RKN resistance mechanisms impact the nematode's post-infection development, delaying or disrupting its life cycle.
Grafting is effective against Fusarium wilt but less so against RKN, though new rootstocks resistant to both pathogens are being developed.

1. Introduction

The Cucurbitaceae family includes economically important crops grown worldwide.
Examples: calabash, cucumber, gourds, luffas, melons, pumpkins, squashes, watermelon, zucchini, and species for medical/ornamental uses.
Worldwide cucurbit production in 2018: Estimated at 234,143,923 tons from 8,315,995 ha.
Root system diseases are mainly caused by soil-borne pathogenic fungi (SBPF) and plant-parasitic nematodes (PPN), which disrupt the plant's vascular system.
Management of soil-borne diseases is shifting from chemical to non-chemical methods.
Alternative control methods include biosolarization, biological control, biopesticides, and cultural management.
- Biosolarization involves soil solarization and organic amendments under plastic films, reducing pathogen populations at temperatures of 40-45°C for 4-6 weeks. However, it can be limited by organic material availability and technical application issues in greenhouses.
- Commercialized biological control agents based on Bacillus, Gliocladium, Pseudomonas, Purpureocillium, and Trichoderma increase cucurbit growth and yield.
- Biopesticides based on essential oils, plant extracts, and microbial metabolites are used against soil-borne diseases, but these alternative methods are generally less effective than chemical soil disinfestation.
Plant resistance is an effective, sustainable, and economic method and a first option in integrated disease management.
The Cucurbitaceae family has a remarkable genetic diversity that can be used to explore sources of resistance or tolerance to key soil-borne pathogens.
Genome sequences of several cucurbit species are now available.
Grafting has been investigated as a management option for SBPF and PPN.
This review examines plant resistance to pathogens co-existing in the rhizosphere, mechanisms involved, and traditional and biotechnological tools for breeding resistance, with an emphasis on grafting.

2. Soil-Borne Diseases in Cucurbitaceous Crops

2.1. Soil-Borne Fungi

In protected cultivation, cucurbits are affected by soil fungi, causing damage to root and crown tissues.
Soil-borne pathogenic fungi and oomycetes (Phytophthora spp., Pythium spp.) can share symptoms such as browning, scars, girdled stem, stunt, loss of root density, wilting, decay, damping-off, and rots of roots and crown.
Vascular wilt diseases are caused by different formae speciales of Fusarium oxysporum, which are host-specific and are the most important SBPF affecting cultivated cucurbits.
Fusarium spp. formae speciales cause asymmetrical brown vascular discoloration.
The continuous use of resistant plants against Fusarium wilt has led to the emergence of new races and formae speciales.
Fusarium oxysporum f. sp. melonis (FOM) is pathogenic to melon and cucumber, with races 0, 1, 2, and 1.2. Race 1.2 strains overcome two dominant resistance genes (Fom-1 and Fom-2).
Fusarium oxysporum f. sp. niveum (FON) races 0, 1, 2, 3 causes vascular wilt in watermelon and squash.
Fusarium oxysporum f. sp. cucumerinum (FOC) affects cucumber, melon, and watermelon with 0, 1, 2, and 3 races.
Other formae speciales include: F. oxysporum f. sp. lagenariae, F. oxysporum f. sp. luffae, F. oxysporum f. sp. momordicae, and F. oxysporum f. sp. benincasae.
Fusarium oxysporum f. sp. radicis-cucumerinum (FORC) is pathogenic to cucumber, melon, watermelon, C. pepo, and L. aegyptiaca and causes damage on roots and mainly the basal stem, but is not a typical vascular pathogen.
Fusarium solani f. sp. cucurbitae: There are two races (race 1 and 2). They can be classified depending on their ability to produce fruit rot.
Pythium spp., Rhizoctonia solani, and Acremonium cucurbitacearum are polyphagous damping-off pathogens that induce water-soaked lesions.
Macrophomina phaseolina and Phomopsis spp. produce pycnidia on lesions and cause necrosis on crown and roots of cucumber, melon, and watermelon adult plants.
Phytophthora capsici and Fusarium solani f. sp. cucurbitae cause damages localized on the stem base on a wide range of greenhouse cucurbits.
Monosporascus spp. are involved in vine decline, which is a syndrome specifically linked to fruit growth and ripening in melon and watermelon. Monosporascus cannonballus produce perithecia on dead roots.
Olpidium bornovanus, which only produces slight root necrosis by itself, causes the vine decline syndrome in combination with Melon necrotic spot virus (MNSV).
Verticillium dahliae directly invade the xylem without root or crown damages.
Each pathogen could especially affect specific plant tissues or plant development stages.
All these SBPF are distributed worldwide and can cause plant death and significant yield losses.
Disease outbreaks occur due to less crop rotation or banning of fungicides, as well as the emergence of more aggressive strains.

2.2. Plant-Parasitic Nematodes

Nematodes associated with cucurbits include Belonolaimus, Criconema, Criconemoides, Dolichodorus, Hemicriconemoides, Hemicycliophora, Helicotylenchus, Heterodera, Hoplolaimus, Longidorus, Meloidogyne, Nacobbus, Paratylenchus, Paratrichodorus, Pratylenchus, Rotylenchus, Rotylenchulus, Tylenchorhynchus, and Xiphinema.
Meloidogyne (root-knot nematodes, RKN) is the most important due to its distribution, damage, and economical importance.
Meloidogyne spp. are polyphagous obligate sedentary endoparasites that disrupt the vascular system of the host plant.
Nematode infection starts when second-stage juveniles (J2) penetrate the roots and induce the formation of feeding sites. The final result is galls in the roots consisting of hyperplasia of the root cortical cells.
Maximum yield losses due to RKN: Estimated at 88% for cucumber, 53% for zucchini, and 35% for watermelon.
Economic losses: About €2.3 million per cropping cycle in 17,500 ha of greenhouse-grown cucurbits in Southern Spain representing 5% of the market value received by farmers.
When assessing RKN resistance, two concepts should be considered: the suitability of the plant to reproduce the nematode (host status) and the damage suffered by the plant due to nematode parasitism (host sensitivity).
The suitability of a host plant for a specific nematode is expressed as the ability of the plant to reproduce the nematode, and it is measured by its reproduction factor (Rf = arc{Pf}{Pi}), that is, the ratio of final population densities at the end of the crop $(Pf)$ to the pre-planting population densities $(Pi)$ .
Susceptible host plants show a Rf > 1, whereas resistant or non-host crops register a Rf < 1.
Cucurbit crops are all hosts to Meloidogyne spp. but differ in host suitability.
Lower Rf is recorded on watermelon and zucchini than on melon, cucumber, and bottle gourd in this order.
There is a large variation in Rf among cucurbits that could be useful for nematode management since less susceptible hosts (low Rf) would reduce the Pf.
A great variation in Rf was found among 15 cucumber cultivars in response to Meloidogyne incognita infection but none was found to be immune or highly resistant.
Cucumber ‘Long Green’ was reported as resistant (Rf < 1), four cultivars as moderately resistant ( $Rf:1–2$ ), and the remaining ones showed several susceptibility levels.
Eight out of 15 melon genotypes were classified as resistant to Meloidogyne javanica (Rf < 1) and promising for use in melon breeding programs, but none was resistant to M. incognita.
Most plant genotypes show different host suitability depending on the infecting RKN species, suggesting that the interaction between the host plant and the nematode species is highly specific.
Therefore, accurate identification of RKN species is necessary for the choice of the cucurbit crop.
Host sensitivity is measured as the damage suffered by the plant when infected by nematodes.
Host plants to which the nematode multiplies but suffer little damage are termed tolerant.
The degree of root galling indicates the pathogenic potential (ability to cause disease) of the nematode in a host and measures the disease severity.

3. Mechanism of Resistance to Soil-Borne Diseases

Passive defense in plants against pathogens begins with the perception of conserved molecular patterns associated with microbes or pathogens (PAMP) by the pathogen recognition receptors (PRR) in plants.
The pathogen-secreted effectors that inactivate the recognition by the plant become avirulent factors (AVR) when they are recognized by resistance proteins (“R” genes), producing a cycle between plant defense responses and the counterattack of the pathogen, described as the zig-zag model.
Plants protect themselves against SBPF and PPN through common mechanisms including hardening of their cell walls, pathogenesis-related (PR) proteins, and production of antimicrobial molecules with an essential role of the plant hormones.

3.1. Soil-Borne Fungi

Non-host resistance to fungi can be produced by lignin accumulation.
Muskmelon and resistant pumpkin (C. maxima) roots infected by A. cucurbitacearum showed a suberized layer in the epidermis.
Additionally, for M. cannonballus, few changes were observed in infected tissues even though pumpkin showed a slight suberin deposition in the epidermis.
Tyloses were formed in the lumen of the xylem vessels of muskmelon and pumpkin, as a response to infection by M. cannonballus and A. cucurbitacearum.
During cucumber infection by FOC, tylose is formed in both resistant and susceptible varieties, but its activation is faster in the resistant ones.
Tylose-mediated response to FOM in melon and FON in watermelon- resistant genotypes has also been described with tylose deposition taking place earlier in resistant varieties.
Plant hormones such as salicylic acid, jasmonic acid, and ethylene are involved in the response of cucumber to R. solani.
The activity of enzymes peroxidase (POX), chitinase, and β-1,3-glucanase is increased during watermelon defense against FON.
The hormonal and enzymatic responses are closely related.
Defense mechanisms can be enhanced by biotic and abiotic agents that promote systemic acquired resistance (SAR, based on PR-proteins, and salicylic acid accumulation) or induce systemic resistance (ISR, independent of salicylic acid, based on jasmonic acid and ethylene).
Biotic agents such as Paenibacillus spp., Pseudomonas spp., Actinoplanes spp., Micromonospora spp., Streptomyces spp. increase chitinase, and glucanase activity and other related PR-proteins in cucumber, improving resistance to R. solani, F. oxysporum f. sp. radicis-cucumerinum, or P. aphanidermatum.
Abiotic agents such as silicate and β-aminobutyric acid derivates can also increase enzymatic activities and phytoalexin accumulation in squash.
Plants could synthesize phytoalexins de novo and accumulate them rapidly at infection sites.
Plant defensins are small, stable, cysteine-rich peptides that could act against fungal pathogens.

3.2. Plant-Parasitic Nematodes

Genetic resistance to root-knot nematodes in cucurbits crops has not been identified, according to our knowledge except for a M. javanica recessive gene (mj) from C. sativus var. hardwickii, but this is not available in commercial cultivars.
Zucchini has shown resistance to M. incognita but not to M. javanica or M. arenaria even though the genetic base of the resistance is presently unknown.
Evidence suggests this resistance is not governed by a major dominant gene because cell necrosis associated with the hypersensitive response does not occur in zucchini, but it may have a quantitative nature.
Mechanisms of resistance restricting RKN parasitism on cucurbits include reduced root invasion rates, delayed nematode development, J2 migration from the roots, increased maleness, and reduced female fecundity.
An increase in root biomass in response to RKN infection was associated with resistance in Cucumis hystrix and Citrullus amarus (ex-C. lanatus var citroides).
Changes in enzymatic activity as a defense response to the RKN attack occur rapidly after nematode infection and the activities of some enzymes such as POX and PAL were greater on resistant C. metuliferus than susceptible bitter gourd or cucumber.

4. Methods of Genetic Control

4.1. Traditional Breeding

Traditional breeding has improved yield and quality of the fruits.
Biotechnological tools have become essential in breeding programs to accelerate any adaptation process.
Traditional breeding includes screening large collections of wild plants to phenotype their resistance response.
Candidates can be used directly as new cultivars or for hybridization and grafting purposes.
Analysis of F1 and F2 and backcrosses obtained by crossing resistant and susceptible genotypes allowed finding the dominant/recessive nature and the number of genes involved in the resistance.
The final objective of these methods is to incorporate these genes into breeding programs for their introgression in commercial or domesticated cultivars.

4.2. Biotechnological Tools

Biotechnological tools have been used for indirect screening for pathogen resistance.
They include the use of molecular markers, omic analysis, mutagenesis, transgenic plants, and, more recently, clustered regularly interspaced short palindromic repeats (CRISPR) systems.
Techniques such as in vitro plant tissue culture could be useful when combined with others.
Transformation mediated by Agrobacterium tumefaciens is a tool for producing whole transgenic plants or pathogens or for CRISPR modifications.

4.2.1. Molecular Markers

In genetics, a molecular marker is a polymorphism in a certain location within the genome that could be associated with specific germplasm or traits.
Molecular markers can lead to a quantitative trait locus (QTL) when they are associated with a phenotypic trait, such as disease resistance.
The importance of markers in breeding, when they are highly associated with a trait of interest, is their use as tools to perform marker-assisted selection breeding (MAS) or to be incorporated into resistant gene introgression programs by taking into account that the markers are often plant germplasm-dependent.
Numerous marker types have been used in cucurbit resistance breeding: single nucleotide polymorphisms (SNP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSR), sequence characterized amplified region (SCAR), and cleaved amplified polymorphic sequences (CAPS), among others.

4.2.2. Omics Analyses

Cucurbitaceae species contain a significantly lower number of nucleotide-binding site-leucine rich repeats (NBS-LRR) genes, closely related to resistance, than other plant species of a similar genome size.
Differential gene expression analysis focusing on single genes or families has been performed as well.
Transcriptome profiling of C. metuliferus infected by M. incognita showed the involvement of hormone biosynthesis and cytoskeleton-related genes in the resistance response as well as many pathogenesis-related genes and RKN effectors.

4.2.3. Mutagenesis and Targeting Induced Local Lesions in Genomes (TILLING)

The natural process of mutation can be accelerated artificially by physical and chemical approaches.
When mutant production and analysis are used in conjunction with large-scale mutation detection like NGS, it is known as TILLING (Targeting Induced Local Lesions in Genomes).
Ecotype TILLING (EcoTILLING) is a similar process but is based on locating mutations in natural germplasm.

4.2.4. Transgenic Plants

There have been some attempts to produce transgenic cucumbers with chitinase gene to increase resistance to pathogens through the incorporation of R proteins, but without much success.

4.2.5. CRISPR

CRISPR (clustered regularly interspaced short palindromic repeats) gene editing is a technique that makes punctual and directed changes in the genome.
The recognition sequence (sgRNA) of the region to be modified can be designed, and the linked endonuclease (CRISPR associated protein, ‘Cas’) cut the target sequence, whose repair will produce the mutation.

4.3. Grafting

Grafting deserves specific comments since this technology has been extensively used in cucurbits to control soil-borne pathogens.
Rootstocks afford increased vigor and yield, even in the absence of pathogens, and provide tolerance to abiotic stresses such as low temperature and salinity.
Rootstocks can include intraspecific selections that exploit specific major resistance genes and interspecific or intergeneric selections that exploit non-host resistance mechanisms or multigenic resistance.

4.3.1. Grafting Cucumber

Cucumber benefits from grafting because it is highly susceptible to Fusarium wilt and RKN.

4.3.2. Grafting Melon

The squash hybrids are effective against all races of FOM, including 1,2 pathotypes and they had no negative effect on yield or fruit quality.

4.3.3. Grafting Watermelon

The squash hybrids are effective against Fusarium wilt due to their non-host resistance to FON.
Lagenaria siceraria is also used as rootstock for watermelon due to its moderate resistance to FON and high compatibility.

4.3.4. Other Possible Rootstocks

Liu et al. found that 12 out of 53 accessions of Cucumis were resistant to FOM and five to M. incognita. Of these, C. pustulatus was resistant to both pathogens and highly compatible with cucumber, melon, and watermelon.

4.3.5. Effect of Grafting on Disease Severity and Crop Yield in Root-Knot Nematode-Infested Soils

The benefit of grafting in promoting crop yield in RKN-infested soils depends primarily on the pre-planting nematode densities in soil and the susceptibility of the crop.

5. Conclusions

Soil-borne diseases are a major threat to cucurbit crops worldwide.
SBPF and RKN constraint profitability of cucumber, melon, watermelon, and squash fruits.
Soil disinfestation with chemicals has collateral environmental and health risks.
Plant resistance is the preferred method to prevent damages and yield losses associated with soil-borne pathogens.
Grafting has proven to be the best solution for specific soil-borne diseases in order to increase pathogen tolerance associated with the more vigorous root system.
New sources of resistance are demanded due to incompatibility issues between rootstocks and scions, market demands, emergence of new pathogen races able to overcome plant resistance, or introduction of new pathogens to the region.
Research has focused on the mechanisms of resistance and breeding tools for resistance to soil-borne diseases of cucurbit crops.
New biotechnological tools are available to complement traditional breeding programs.