Main methods of spread and transmission of viruses in crop production

4 cm | SM, _ 3% > y | — `, ut u 3 and 9.1.1. Dissemination and conservation of viruses

Viruses causing plant diseases can spread in various ways. Transmission is possible through mechanical contact, i.e., during mutually damaging contact between parts of healthy and diseased plants. This occurs during the contact of above-ground or underground plant parts in dense planting, as well as during plant care processes (pinching off suckers, cutting flowers, harvesting fruits, etc.).

A significant number of viruses are seed-transmitted. These include legume viruses (bean mosaic, soybean mosaic), tomato mosaic (ToMV), and cucumber green mottle mosaic virus (CGMMV). Moreover, a distinction is made between surface and internal seed infection. Viruses can also enter seeds via pollen during pollination, but this transmission method has been reliably proven only for a few viruses (mainly of pome and stone fruits).

For vegetatively propagated crops, the main way of virus dissemination is nursery plant material. During grafting (transplantation), the spread of viral diseases occurs. All known phytopathogenic viruses are transmitted by this method.

Single viruses (tobacco mosaic virus, tobacco necrosis virus) can be transmitted via plant residues, soil, or hydroponic solution.

Many phytopathogenic viruses are spread by invertebrates with piercing-sucking mouthparts, primarily aphids, as well as leafhoppers, thrips, bugs, whiteflies, beetles, mites, and nematodes. The mechanism of virus spread by vectors varies, and depending on transmission characteristics, viruses are divided into persistent and non-persistent.

Vectors are generally specialized species, most often aphids, less frequently leafhoppers, thrips, bugs, and mites. Soil nematodes parasitizing plant roots are often vectors for a number of fruit, berry, and ornamental crop viruses. There is data on the spread of some viruses by soil fungi. It has been established that tobacco and cucumber necrosis viruses are transmitted by zoospores of Olpidium brassicae, and potato virus X (PVX) by the potato wart pathogen (Synchytrium endobioticum).

Non-persistent viruses spread mainly due to the attachment of viral particles to the surface of the stylet of the piercing-sucking apparatus. Non-persistent viruses can be transmitted by a vector within a limited period of time, often no more than an hour, after which the transmission rate decreases. Moreover, a vector feeding on a diseased plant acquires such a virus, i.e., becomes viruliferous very quickly — within the first seconds, rarely minutes. After the vector molts, infectivity is lost. Non-persistent viruses include potato virus Y (PVY), pea mosaic virus, common cucumber mosaic, and others.

With the persistent method of dissemination, the vector, while feeding on an infected plant, becomes viruliferous after several hours or days and retains infectivity for more than 100 hours, and sometimes for its entire life. This feature is maintained regardless of molting.

Entering the digestive system of the insect vector with plant sap, viruses reach the hemolymph, and from there to the salivary glands, where they can even multiply (propagative viruses). Some viruses (tomato spotted wilt virus, squash mosaic, potato leafroll) are capable of transovarial transmission, i.e., subsequent generations of insects become infected.

There is also a semi-persistent method of transmission. In this case, the vector retains viruliferous status for 10 to 100 hours. This is how PMV (potato M virus) is transmitted.

Most viruses overwinter in the living parts of perennial cultivated plants or weeds (in particular, in root crops, roots, bulbs, cuttings, etc.). Their transmission from year to year leads to a sharp decrease in plant productivity and cultivar degeneration. Some viruses persist in the body of vectors, others are stored on the surface or inside seeds.

The most resistant viruses, such as tobacco mosaic virus (ToMV) and green cucumber mosaic (CGMMV), are capable of persisting for a long time on plant residues, in the substrate, and in recycled irrigation water of drip systems.

A few examples:

• Tobacco mosaic virus (TMV, ToMV) — transmitted via tomato seeds. In addition, plant residues with TMV in the soil, where the virus persists for a long time due to its stability, serve as a source of infection. The pathogen can also persist on greenhouse structures, work clothing, and tools. It reserves on many species of cultivated and wild plants growing near greenhouses.
• Potato virus X (PVX) — can be transmitted via seeds. Potato tubers and packaging brought into greenhouses can also be a source of infection.
• Cucumber mosaic virus (CMV) — transmitted by many species of sucking pests: aphids, thrips, whiteflies. Various species of perennial and annual plants, including cucumber, can serve as reservoirs.
• Potato leafroll virus (PLRV). Various aphid species are known as its vectors. It has been established that this virus is transmitted via tomato seeds, although previously mechanical and seed-based transmission for PLRV was unknown. The virus infects cultivated plants, predominantly annuals, in temperate zones, and is primarily spread on potatoes.

In nature, those virus strains survive that reproduce intensively but, at the same time, do not cause serious damage to their hosts that could lead to the death of the plant. As a result, the infected plant becomes a reservoir of the virus, which is easily spread by vectors among the plantings.

At a young age, plants are most susceptible to viral infections and, at the same time, most attractive to pests, including disease vectors. This feature must be taken into account when determining the sowing dates for crops and carrying out protective measures against pests. In a number of cases, this makes it possible to prevent the development of viroses.

As they grow and age, plants acquire resistance. The rate of disease spread in plantings of old plants decreases not only due to their greater resistance, but also because the reproduction and systemic spread of viruses occur more slowly.

Plants grown according to all the rules of agrotechnology are more sensitive to infection.

The rate of virus spread among plantings depends on the biological characteristics of the vectors. Thus, it has been established that winged females of the greenhouse aphid prefer plants with chlorotic coloration. On such plants, the viability of the pest increases:

which, ultimately, leads to a greater increase in the number of vectors. 1.1.2. Diagnostic methods viral diseases

Visual diagnostics. Although in a number of cases it is possible to reliably establish the viral nature of a disease by external signs (for example, ring or linear chlorotic patterns on young leaves), their identification is difficult due to the asymptomatic (latent) nature of disease development.

Symptomatology depends on the state of the organism, the aggressiveness of the pathogen strain, external conditions, and the duration of the virus's presence in the host cells. For example, symptoms of the disease are clearly expressed in plants that have grown in bright light and at a moderate temperature; at high temperatures and insufficient lighting, the symptomatology of the disease may not be expressed at all.

Clearing of the veins of the youngest leaves is often one of the first signs of a systemic viral infection. The veins become yellow and translucent. Leaves formed later may be:

• mosaic;
• mottled;
• completely yellow (chlorotic).

The incubation period of a disease depends on the type of virus, the host plant, and environmental conditions, but, in any case, it lasts several days or weeks.

If the first symptoms of virosis are noticeable on transplants, then the source of infection was either in the seeds, or the infection occurred through a vector. If infected plants are located in the crop completely randomly or only in rows planted from one batch, this indicates that, most likely, the planting material was infected. If diseased plants are found in individual parts of a field, and the appearance of infected areas is associated with soil differences, there are all grounds to assume the spread of viruses through the soil.

I would also like to note that there are symptoms resembling viral infections but caused by other reasons. Similar symptoms of damage are exhibited by phytoplasmas and some bacteria.

Point necroses caused by plant damage by aphids and mites are also often confused with viroses; for example, the potato aphid causes curling, deformation, and local chlorosis of cucumber leaves. The appearance of symptoms similar to a viral infection is often caused by common mineral nutrition disorders (for example, associated with iron deficiency). Various deformations of organs can be caused by hormonal herbicides.

Thus, accurate identification based on external signs of viral infection is impossible; an unequivocal answer can only be obtained using instrumental methods (enzyme-linked immunosorbent assay, PCR analysis, etc.).

The indicator plant method is a widely used method for diagnosing viral diseases and identifying viruses. It is based on the use of test (indicator) plants that produce clear, often strictly specific symptoms in relation to a certain type of virus. Infection of herbaceous indicator plants is carried out by mechanical inoculation with sap. The infection manifests itself in the form of local necroses, less often by a systemic reaction (change in color, growth inhibition). For the tomato aspermy virus, young tobacco plants (Nicotiana glutinosa) can be used as an indicator, and for the diagnosis of potato X-virus — globe amaranth (Gomphrena globosa). In some cases, individual isolated leaves of indicator plants can be used for infection. Non-sap-transmissible viruses are transferred to indicator plants by grafting; for example, for the diagnosis of strawberry viruses, grafting of a leaf petiole onto indicator clones of wild strawberry (Fragaria vesca) is used. Rarely, insect vectors and the parasitic plant dodder are used to transmit viruses.

Serological diagnosis. If a purified plant virus preparation is introduced into the blood of a warm-blooded livestock animal, the animal's immune system will begin to produce specific antibodies that bind to the foreign protein (antigen) in response. As a result of the reaction, a precipitate (serum) is formed, which is visible to the naked eye or under a microscope.

Diagnostic methods: how to detect a viral infection

Laboratory and rapid methods are used for accurate virus identification in crop production. They allow for the detection of a pathogen at early stages, determining its species and even strain. The choice of method depends on the virus structure, its concentration in tissues, and the available equipment.

For rapid assessment in field laboratories, simple serological tests are used. The droplet method allows mixing plant sap with antiserum on a glass slide and evaluating the reaction visually or under a microscope within a few minutes. The adsorption method increases clarity: antibodies are pre-bound to latex or another coarse-dispersed material, which, upon contact with the virus, produces a well-noticeable agglutination of the entire complex.

When working with complex or small viruses, gel diffusion methods are used. In double diffusion, the antiserum and purified sap move towards each other in agar, forming precipitation lines. In radial immunodiffusion, the antiserum is introduced directly into the gel, and the wells are filled with sap, resulting in ring-shaped precipitates in case of a positive reaction.

The most accurate quantitative method remains enzyme-linked immunosorbent assay (ELISA). This method is based on the use of specific enzyme labels — phosphatase or peroxidase.

1. Antiserum and purified plant sap are added to the wells of a polymer microplate for the first immune reaction to occur.
2. A solution of enzymes (phosphatase or peroxidase) conjugated with antibodies is applied to the resulting fixed antigen-antibody complex.
3. Enzyme-labeled antibodies layer onto the antigen molecule determinants, ensuring a second immune reaction.
4. After the addition of the appropriate enzyme substrate, a catalytic cleavage reaction is triggered, which is recorded using a photometer based on the color reaction.

For in-depth structural research, electron microscopy is used. A transmission microscope allows for the examination of the shape, size, and structure of the virus in ultrathin sections of affected plant parts. The immuno-electron microscopy method helps to conduct serial analyses, detecting viral particles with antibodies layered on them directly in the sap.

Molecular analysis methods are based on studying nucleic acids of pathogens. The DNA probe method recognizes specific regions of viral RNA based on the principle of complementarity, which allows for distinguishing between groups, species, and strains of viruses. In molecular biological analysis using the polymerase chain reaction (PCR), enzymes repeatedly copy species-specific RNA regions, after which they are detected by electrophoresis or immunofluorescence.

• Current intensity for separating nucleic acids in gel — 3 mA
• Current intensity for separating virus proteins in gel — 6 mA
• Amplification of RNA fragments during PCR — millions of times

Specialized methods include the analysis of cellular structures under a light microscope (inclusion method). The accumulation of viruses in the cells of the epidermis or leaf hairs leads to the formation of clusters (Ivanovsky crystals) of a characteristic shape. Needle-like and hexagonal crystals are characteristic of the tobacco mosaic virus, while spherical amorphous bodies are typical for the potato X-virus. Chemical analytical diagnostic methods are also used to detect cucumber green mottle mosaic and potato leafroll.

Protection strategy: why chemicals are powerless and how an agronomist should act

Viruses are constantly present in plant organisms. By themselves, they may not cause the death of the host, but when stress conditions arise, their harmfulness increases sharply. Plants attempt to defend themselves, reacting to the infection with the appearance of necrosis, mosaics, and organ deformations, which leads to a decrease in yield and deterioration of product quality.

Due to the lack of effective therapeutic agents, the protection of sowings and plantings is exclusively preventative. The control of viral diseases in field conditions is based on preventing infection. In practice, agronomists use plant vaccination with weakly pathogenic viral strains and slow down the development of viral epiphytotics using a complex of agrotechnical practices.

• During vegetative propagation, periodic roguing of mother plant plantings is carried out. This method is effective for controlling viruses that have clearly distinguishable symptoms.
• Inspection of young plants and culling of infected plants to destroy primary sources of infection. Thorough inspection of plants during the emergence, beginning of flowering, and beginning of fruiting stages.
• Thermotherapy allows for a sharp reduction in infection rates, and sometimes entirely rids plants of viruses. This method can be used both for disinfecting vegetative organs and for controlling infection within seed. Temperature regimes are strictly specific and are discussed below in the relevant sections.
• Using the apical meristem culture method allows for the elimination of most viral pathogens. The best healing effect from viral infections is obtained by combining the apical meristem culture method with preliminary thermotherapy or chemotherapy, in which antiviral additives (glycoproteins, polysaccharides, nucleic acids, higher plant antibiotics) are introduced into the nutrient medium for meristem cultivation, or the initial meristem donor plants are treated with them.
• Control of virus reservoir plants and vectors of infection. Reducing the virus reservoir in environmental objects (in seed and in the plants themselves, control of infection vectors).
• Use of immune cultivars and hybrids. In this case, breeding work should be carried out not only for resistance to the virus, but, preferably, to its vector as well. Of no less importance is the development of tolerant (hardy) cultivars, in which the systemic spread of viruses is limited and their concentration is reduced. Tolerance often leads to asymptomatic disease progression, while plant productivity is practically not reduced.
• Stimulating non-specific immunity in plants using elicitors (inducers).
• Pre-immunization, or vaccination. It is known that virulent strains do not cause disease symptoms if the plant has previously been infected with a weakly pathogenic or avirulent strain of the virus.

Similar vaccination was previously used to protect TMV-susceptible cultivars and hybrids of tomato. However, on the whole, pre-immunization has not become widely used due to the possibility of pathogen mutation, increased harmfulness upon co-infection with other pathogens, and a number of other reasons. However, in recent years, good vaccines have been obtained not only against TMV but also against cucumber green mottle mosaic virus.

Changing the plant genome by incorporating new resistance genes obtained from donors. When a gene responsible for the synthesis of the tobacco mosaic virus coat protein is introduced into cells, it induces resistance in tobacco to this disease. For instance, transgenic zucchini carrying genes for the viral coats of zucchini yellow mosaic and watermelon mosaic showed no symptoms of viral infection, whereas control plants and transgenic plants with only one gene had clear damage.

Field trials conducted on virus-resistant tomato, potato, and many other crops obtained using this approach have demonstrated its effectiveness and the prospects for further research in this field.

State and on-farm quarantine. When importing plants, the certification must confirm that the material does not contain quarantine pests. The effectiveness of external and internal quarantine measures largely depends on the reliability and speed of virus identification methods.

Organizational and management measures include disinfection of cutting tools and implements in a solution of formalin, potassium permanganate, alcohol, or their heat treatment, as many economically significant viruses are transmitted by contact; working in change-of-shift footwear and clothing; placing disinfectant mats at the greenhouse entrance; and regular visual inspection of plants.

Alleviation of disease symptoms by maintaining optimal crop growing conditions, including mineral nutrition. During the development of an epiphytotic, plants are sprayed with solutions of trace elements, phosphorus, and potassium fertilizers, which stimulate early plant ripening and, consequently, the onset of age-related resistance.

The latter three methods together form the basis of so-called preventive measures. 1.2. Viroids

This is a relatively new group of virus-like phytopathogens. In fact, viroids consist of a single cyclic RNA molecule that is capable of replicating in a plant using the host plant's biosynthetic mechanisms. Viroid RNA consists of 250-400 nucleotides and has a unique structure. It is a circular molecule with a high percentage of base pairing, as a result of which viroids are resistant to high temperatures (over 100°) and chemical substances (phenol, alcohols, etc.). Viroid diseases were known previously, but they were considered viral or as diseases of undetermined etiology.

Viroids are highly infectious pathogens capable of mechanical transmission, as well as transmission via grafting, pollen, seeds, insects, and parasitic plants such as dodder. They primarily infect crops of the dicotyledonous class. There are virtually no effective control measures for the disease. This group of pathogens is particularly dangerous for perennial and vegetatively propagated plants.

The spread of viroid diseases is facilitated by the difficulty of detecting the pathogen in samples of initial planting material, including that imported from abroad. In breeding and genetic work, transmission of viroids via pollen and seeds is possible. These diseases cause damage to citrus, chrysanthemum, tomato, cucumber, and eggplant.

Tomato can be infected by potato spindle tuber viroid (PSTVd), citrus exocortis viroid, as well as tomato planta macho and apical stunt viroids. In Holland, and later in other countries, a viroid disease of cucumber was noted—pale fruit, which becomes noticeably manifest during summer months on long-fruited parthenocarpic hybrids. In recent years, latent infection of eggplant, beans, and cucumber by citrus exocortis and hop stunt viroids is increasingly being detected. In general, information on the distribution and harmfulness of viroid diseases on vegetable crops is still scarce.

Viroid diagnostics are conducted using indicator plants, as well as methods for identifying their nucleic acids: polyacrylamide gel electrophoresis (PAGE), molecular hybridization, and polymerase chain reaction (PCR).

The main focus of protection against viroids is preventive measures: the use of healthy planting material, tool disinfection, culling of infected plants, etc. It is important to consider that plants infected with viroids cannot be cleared of the infection using apical meristem methods.

Bacteria and phytoplasmas belong to prokaryotes, i.e., the cells of these organisms lack a true nucleus. A circular DNA molecule is located in a specific area of the cell called the nucleoid. In terms of cell wall structure, prokaryotes differ significantly from nucleated organisms, known as eukaryotes. The cell walls of prokaryotes lack chitin and cellulose, which are characteristic of fungal or plant cells. The supporting framework of their cell walls is formed by the glycopeptide murein.

Bacteria are single-celled organisms. The length of a bacterial cell is 1-3 µm, and the width is 0.3-0.6 µm. Almost all phytopathogenic bacteria (PPB) have a rod-like shape (with the exception of Streptomyces, which have a filamentous structure). Most PPB are motile due to the presence of flagella; there are few non-motile forms. Bacteria may have one or more flagella. Depending on the number and arrangement of flagella, all motile bacteria are classified as monotrichous—with one polar flagellum, lophotrichous—with a bundle of flagella at one end of the cell, and peritrichous—with flagella located over the entire surface of the cell. For most motile PPB, the flagella are polar; peritrichous arrangement is rarer.

The nucleoid of a bacterial cell consists of DNA and is distributed in the cytoplasm in the form of small granules, not separated from the cytoplasm by a membrane. The bacterial cell is surrounded by a relatively thick multilayered envelope—the cell wall, the inner supporting layer of which gives the bacterium a certain shape.

Under unfavorable environmental conditions, for example, under the influence of antibiotics, some species form so-called L-forms without cell walls, which, however, can restore their original structure under certain conditions.

The envelope of some PPB is covered with a thin mucous layer of exopolysaccharides, which has the ability to swell, resulting in the formation of a capsule. The mucous capsule is of great importance for the survival of bacteria in unfavorable conditions; they become resistant to the effects of sunlight, chemicals, and other factors. In humid weather, bacterial cells multiply on the surface of plant organs and accumulate in the form of clusters of mucus or exudate.

An important method for the identification of bacteria is based on the features of their cell wall structure—Gram staining. The essence of this method is the staining of fixed bacterial cells with a solution of crystal violet and Gram's iodine, followed by decolorization with ethyl alcohol, after which in some species, the dye is washed out of the walls and they are decolorized (Gram-negative bacteria), while in others, the dye is firmly bound by the cell wall. As a result, the cells turn blue (Gram-positive bacteria). Almost all PPB are Gram-negative; only species of the genera Clavibacter and Streptomyces give a positive reaction.

PPB are capable of multiplying at 5-10°, the optimal temperature is 25-30°, and with an increase in temperature to 33-40°, bacterial reproduction ceases. For active life activity, PPB require a neutral or slightly alkaline environment, which distinguishes them from fungi, for which an acidic environment is favorable for growth.