Methodology for assessing root-knot nematode infestation in greenhouse crops

How to assess infestation: mapping methodology and yield loss calculation

Assessment of infestation of greenhouse crops root-knot nematodes is carried out at the end of the growing season or when removing plant residues after a production cycle. To obtain reliable data, it is necessary to correctly calculate the average damage score of the root system. This will allow for timely adjustment of the protection scheme and forecasting future yield.

• Sampling for examination — at least 1500 roots
• Number of plants in one infestation focus — 20–30 pcs. consecutively
• Correction of the score during excavation — +0.5 points
• Period of yield loss manifestation in tomatoes — 4th–5th month of the growing season

To minimize errors when mapping the infestation level, follow this sequence of actions:

1. Conduct the examination during the removal of plant residues after a production cycle or at the very end of the growing season.
2. In each infestation focus, examine the root system of 20–30 plants in a row. In total, at least 1500 roots must be analyzed for the assessment.
3. When examining, mentally increase the degree of damage by 0.5 points. When pulling out plants, some galls are inevitably torn off and remain in the soil, not being included in the analysis.

When assessing tomatoes, it is important to consider the peculiarities of their development. On resistant cultivars and hybrids, due to local loss of resistance, 2–4 large galls may form, while 90% of the roots remain healthy. Such damage is assessed not by the size of the galls, but by the overall percentage of infestation — no more than 1–2 points. Tomatoes are more resilient than cucumbers: a similar level of yield loss is reached on them not in the 3rd–4th, but in the 4th–5th month from the start of infestation or planting of transplants.

The average infestation score (P) is calculated using the formula:

P = (a × 1 + b × 2 + c × 3 + d × 4) / M

Where:

• M — total number of analyzed plants;
• a, b, c, d — number of plants with an infestation score of 1, 2, 3, and 4, respectively.

The value of the average score is directly related to yield losses. The table shows the dependency data for cucumbers for the first four months of the growing season.

Average infestation score	Yield loss, % of control	Effect on the crop
1	Insignificant (cannot be reliably determined)	Even a high number of point galls on the greater part of the root does not cause tangible losses.
2	10–15 % (higher under unfavorable conditions)	Yield losses become tangible for the farm.
3	15–30 %	Significant suppression of plants and drop in productivity.
4	30–70 %	30% of losses occur at the end of the 3rd month, and 70% — at the end of the 4th month after planting transplants.

Resistant tomato hybrids: how to maintain their effectiveness

Using resistant tomato hybrids is a mandatory element of plant protection in infested soil. Their resistance is based on the Mi gene, which inhibits the formation of giant feeding cells in the roots when exposed to larval enzymes. By penetrating the root, the nematode larva cannot feed and grow properly. Under optimal conditions, such hybrids are not inferior to conventional ones in yield and reduce the invasion load on the soil.

Planting nematode-resistant tomatoes in the second production cycle (after cucumbers) does not make sense. During this period, the temperature in greenhouses is too high, and the soil is oversaturated with pest larvae. The hybrids will quickly lose their resistance, which will lead to yield loss and an increase in the nematode population.

A proper strategy for using resistant hybrids is built according to the following scheme:

• Cultivation exclusively in the first (prolonged) production cycle;
• Preliminary implementation of a full complex of anti-nematode measures before planting transplants;
• Strict adherence to the air temperature regime (no higher than 28 °C) during the initial period of the growing season.

At a low level of invasion, resistant tomato hybrids are capable of effectively suppressing the development of the parasite. Even if individual specimens overcome the immune barrier, the plant triggers a mechanism for their sterilization.

The defensive reaction of a resistant plant is expressed in the disruption of the pest's life cycle:

• death of penetrated larvae;
• prolonging their development process from 18–20 to 50–60 days;
• significant reduction in the egg productivity of developed females;
• appearance of non-viable eggs;
• shifting the sex ratio of the offspring towards the formation of males.

In actual production conditions, tomato resistance can be completely lost. There are three main risk factors capable of neutralizing the protective properties of hybrids.

Risk factor	Threshold value
High air temperature	Over 30 °C
Infection pressure	5 or more larvae per 10 g of soil
Presence of nematode species	Complex of species, including the northern root-knot nematode

During the summer in greenhouses of the Central region of the Russian Federation, air temperatures above 30 °C can persist for a month or longer. At the same time, the actual infection pressure in the soil of domestic greenhouse complexes often exceeds 25 larvae per 10 g of soil. Massive simultaneous introduction of parasites leads to a high concentration of their enzymes in the root vascular system, causing the plant to lose its resistance. Therefore, it is most effective to plant resistant hybrids in the first rotation and only after performing anti-nematode treatments.

Cucumber roots grow to a great depth, become infested by the surviving nematodes, and resume the epiphytotic process. There are no fully resistant cucumber hybrids, but there are hardy hybrids for greenhouses (e.g., F1 Typhoon) capable of maintaining yield even with a high level of soil infestation.

For tomato, breeding companies De Ruiter Seeds, Rijk Zwaan, Enza Zaden, Gavrish, and Semko-Junior offer a wide range of F1 hybrids with genetic resistance to root-knot nematodes. These include: Faraon, Shulga, Figaro, Shuttle, Evpator, Valet, Tali-Tsa, Semko-99, Semko-101, Tolstyachok, Wunderkind, Otlichnik, Unikum, Kal-roma, Fancy, Streza, Strauss, Olya, Kupets, Flamingo, Viscount, Annabelle, Bentse, Enna, Nagano, Fontana, Baldo, Kilian, Muril, Preti, Gabor, Romatos.

Migratory nematodes of the genus Pratylenchus in protected ground

Representatives of the genus Pratylenchus (including species P. penetrans, P. vulnus, P. crenatus) are dangerous endoparasites of the root system. Unlike root-knot nematodes, these small worm-like parasites do not cause gall formation but continuously migrate inside the root, destroying cells and conductive tissues.

In greenhouses, Pratylenchus infect almost all ornamental crops, including roses, chrysanthemums, carnations, violets, and lilies. External symptoms of infestation are manifested in plant wilting and leaf chlorosis. By damaging the roots, nematodes also facilitate the penetration of secondary infections, acting as vectors for Fusarium root rots.

• Life cycle — about 1 month
• Fecundity per female — 80–200 eggs
• Maximum density — up to 10,000 individuals per 1 g of roots

The morphology of Pratylenchus is characterized by identical sizes of males and females: body length is 320–800 µm, width is 15–30 µm, vulva position is 70–86%, stylet length is 12–18 µm, and spicule length in males is 12–15 µm. The worm cuticle is annulated, the stylet is well-developed with rounded knobs at the base, the female tail is relatively short with a rounded end, and the male tail has a transparent bursa.

Protection measures against these nematodes have not been specifically developed. It is recommended to use generally accepted anti-nematode measures: avoidance of monoculture, use of only nematode-free soil. Fig. 68. MORPHOLOGY OF Pratylenchus. Legend: A — female; B, C — head ends; D — male tail end; E — female tail end. 1.5.5. General methods of plant protection against root-knot nematodes 1. Greenhouse complexes should not have any contact with botanical gardens and private orangeries regardless of whether they have root-knot nematodes, and should not bring in any crops with a root system (including scallions) without professional inspection by phytohelminthologists. 2. When growing vegetable and ornamental plants simultaneously in the greenhouses of one complex, the movement of personnel, machinery, tools, and agricultural implements between them should be limited. If part of the complex is infested with root-knot nematodes, strictly follow accepted quarantine measures, the main ones being complete removal of soil from hand tools, wheels, and agricultural machinery, disinfection of personnel footwear, spatial isolation, and thorough preparation of substrates and soils for crop rotation. 1.6. Non-infectious disorders Most non-infectious diseases have symptoms related to changes in the color or shape of certain plant organs. For example, leaf chlorosis caused by nitrogen or iron deficiency. Several types of physiological diseases are distinguished, which can be systematized by the nature of the impact of external factors on the plant: 1. Disruption of mineral nutrition — rarely leads to total plant death; more often, developmental delay, reduced yield, and impaired ornamental value (for flower crops) are observed. Soil or substrate salinization is the most dangerous. This can cause the appearance of numerous leaf and flower burns, fruit set drop, and plant death. 2. Violations of growing conditions (temperature and relative humidity, photoperiod duration, or light intensity) — cause disturbances appearing as burns, necroses, spotting, and color changes, but lead to plant death only at extreme values. It is interesting that the external symptoms of infestation of the same plant species at different temperature values are visually similar. For different plants, the response to the same environmental impact can trigger an opposite reaction. Thus, with a lack of light, eggplant fruits lighten, but this same symptom appears on tomatoes under increased solar radiation. 3. Chemical phytotoxicity — is caused by high concentrations of various substances, including pesticides. It manifests as burns, curling of shoots, or deformation of leaves, flowers, and fruits. In case of severe poisoning, plants die. Fig. 69. TOMATO FRUIT BURN CAUSED BY FROST. 4. Greenhouse air pollution causes poisoning (most often by sulfur dioxide, ozone, chlorine, nitrogen dioxide), resulting in plants usually dying or being covered with multiple burns on leaves, flowers, and young shoots. 5. Mechanical damage, as a rule, delays plant development. Damage to roots and the stem base, which often occurs at the moment of transplanting seedlings or when planting plants in a permanent location, is the most dangerous. Plants tied to a trellis suffer from tugging, which leads to temporary wilting or their complete death. 6. Teratoplasmoses — genetic disorders. The causes of organ developmental disorders are often genetically determined or are the result of the traumatic impact of environmental factors on apical and lateral meristematic cells. Fig. 70. MARGINAL LEAF BURN CAUSED BY INCREASED PESTICIDE CONCENTRATION.

“ > O — y % e ” „” # v Chao 4 b. _ This group of disorders is characterized by specific symptoms caused by the traumatic impact of environmental factors on plants. Developmental disorders of organs (teratoplasmoses) have several forms, the description of which is beyond the scope of this publication Fig. 71. FUSION OF TWO CUCUMBER FRUITS 4 ^^ 1.7. Development of epiphytotics in greenhouses An epiphytotic is a mass disease of plants developing under the influence of disease-causing microorganisms in a limited area over a certain period of time. Any epidemic is a sharp disturbance of the balance inherent in natural communities and is subject to certain patterns. The development of epiphytotics is possible when three conditions are combined simultaneously: a) if a large reserve of disease pathogens has accumulated in a limited area; b) if cultivars susceptible to the pathogen are grown in this area; c) there are optimal environmental conditions for the development of the disease. Some factors can weaken the epiphytotic process, while others can strengthen it. Thus, the intensity of infection decreases if the plants are resistant to races of the pathogen and increases if several virulent races of the pathogen, capable of overcoming resistance, accumulate in the greenhouse. The epiphytotic ceases under the influence of agrotechnical measures and the correct application of plant protection products, both against the pathogen itself and against its vectors.

Any epiphytotic has three stages: preparatory; the epiphytotic itself; and subsiding. During the first stage, the infectious agent accumulates. It can last for quite a long time; for diseases of a monocyclic nature, it can take several years. In the second stage, mass infestation of plants is observed, often ending in their death. In the final stage, the intensity of disease development gradually decreases, which is due either to the biological characteristics of the pathogen or to protective measures (Stroykov, Shkalikov, 1998).

How to stop an infection outbreak in a greenhouse

Monoculture, intensive mineral nutrition, and a reliance on high-yield but non-resistant hybrids often lead to uncontrolled outbreaks of infection (epiphytotics) in protected ground. To suppress an incipient disease, the agronomist must act quickly and comprehensively. Timely adjustment of growing conditions allows for curbing the development of the pathogen and saving the harvest.

1. Sharply change the microclimate parameters — temperature, air humidity, and substrate acidity — to make the environment unfavorable for the pathogen.
2. Eliminate the conditions for mass infection: destroy vector organisms and dry out plants to stop the movement of zoospores.
3. Apply immunostimulants to increase the natural protection of plants and use the entire available complex of plant protection products.
4. Stimulate the recovery of vegetative mass with the help of additional top dressing, growth regulators, increased irrigation, and supplemental lighting.
5. Remove severely affected plants and plant debris that serve as a source of infection, and apply curative preparations to combat latent forms of diseases.

To prevent epiphytotics, it is important to activate the activity of natural regulators in the agrobiocenosis. During cutting rooting, treatment with strains of Pseudomonas fluorescens bacteria (163 and AR 33) and Pseudomonas aureofaciens shows high effectiveness. This drastically changes the soil biocenosis in the rhizosphere: the number of parasitic nematodes, fungi, and bacteria decreases, and beneficial microflora (actinomycetes, Trichoderma, microbe-phagous nematodes, and predatory invertebrates) begin to actively multiply.

• Increase in beneficial microflora in the rhizosphere — 2–5 times
• Increase in the yield of rooted cuttings after treatment — 1.4–20.0 times
• Proportion of diseases recurring through infected seed — up to 50%

Cucumber growing conditions and viral risks

Cucumber occupies the largest areas in glass and film greenhouses due to its early maturity and relatively low light requirements. Traditionally, it is grown on organic-rich peat soils; however, the area under low-volume crop cultivation on mineral substrates with drip irrigation is currently expanding actively. The root system of the cucumber is extremely sensitive to air and water regimes, so the crop requires loose, well-drained substrates.

Cucumber growing parameter	Technological standard
Air temperature during the day	22–27 °C
Air temperature at night	17–19 °C
Temperature in the root zone	18–20 °C

Both bee-pollinated and parthenocarpic hybrids are grown in greenhouses. The use of bees imposes strict restrictions on the use of chemical plant protection products, which complicates the fight against pathogens. The greatest danger to the crop is posed by the Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), and Tobacco necrosis virus (TNV).

Of particular danger is the green mottle, or English cucumber mosaic (causative agent — CGMMV). The virus primarily affects young plants, causing leaf deformation (especially in winter months), growth cessation, and premature death of the plantings.

Cucumber green mottle mosaic virus (CGMMV) belongs to the RNA-containing tobamoviruses and causes systemic infection of cucurbit crops. It affects only cucumber, melon, and watermelon, but is not found on pumpkin or squash. In this respect, it differs from the highly specialized parasite Cucumber mosaic virus. The disease significantly reduces productivity: the number of female flowers decreases on infected plants, and fruits develop slowly, become deformed, and lose their marketability.

• Temperature for symptom manifestation — 30 °C
• Manifestation period for seed-borne infection — 14 days
• Epiphytotic development period — 40 days
• Manifestation period for soil-borne infection — 20–30 days
• Persistence in dry leaves — 1 year

Disease symptoms and diagnostic methods

The first symptoms usually appear on young leaves 20–30 days after transplanting, especially when the air temperature rises. The leaf blade becomes wrinkled and reduced. The most pathogenic strain, CGMMV 24, causes white mosaic: first, clearings and yellow star-shaped rings appear along the veins, which then merge and completely bleach the leaf and fruits.

The infection spreads via plant residues, seed, and through the soil, although its specific vector in the soil has not been established. From the point of entry, the virus quickly moves through the vessels into the roots, stems, and leaves. In a greenhouse, the pathogen is easily transmitted by contact with the sap of infected plants during pinching, tying, and harvesting. In hydroponic systems, virus transmission through a shared nutrient solution is possible.

To prevent the introduction of infection into the greenhouse, seed is checked before sowing. A biological assessment method is used for this. The procedure is performed in several stages:

1. Homogenize 10 cucumber seeds in 5 ml of 0.03 M phosphate buffer with a pH of 7.0.
2. Mix the resulting homogenate with a fine fraction of quartz sand.
3. Rub this mixture onto 10 healthy cucumber seedlings.
4. Observe the plants: if the virus is present, mosaic symptoms will appear on the leaves in 8–10 days.

Serological diagnostic methods can also be used for analysis. If the virus is detected, the seed lot is rejected. Seed crops must be located away from other cucurbit crops, sucking pests must be eliminated in a timely manner, and a high level of agricultural practice must be maintained.

To support immunity and reduce the harmful effects of the virus in ground-based greenhouses, special agricultural practices are applied. An optimal mineral nutrition regime helps plants resist the disease. Agronomists also use the following technical solutions:

• lowering the stems to develop an additional root system;
• double top dressing of plants with an aqueous infusion of chicken manure (in a 1:12 ratio) with an interval of 10 days.