PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART I. MICROBIAL BIOLOGY

CHAPTER 3. MICROFLORA OF HERBAL MEDICINAL RAW MATERIALS, PHYTOPATHOGENIC MICROORGANISMS

Medicinal plants harbor a wide spectrum of microorganisms possessing diverse specific properties and exhibiting significant qualitative variations [14]. Basically, the microbial load of plants results from a combination of biotic and abiotic environmental influences, alongside microbial contaminants easily transmitted via air and soil. As illustrated in Fig. 30, plants comprise a subterranean part (roots) and an aerial part, a factor that must be taken into account when analyzing their microbial contamination. The subterranean (rhizospheric) part resides in the soil and is in continuous contact with soil microorganisms (Fungi, actinomycetes, Bacteria), Viruses, and phages, which can either penetrate the roots or colonize the ROOT surface. The aerial part of plants is in constant contact with microorganisms that may settle from dust and Water droplets. The COMPOSITION OF THE airborne microflora can fluctuate periodically with changes in wind direction and is also dependent on the proximity of industrial facilities. When manufacturing medicinal products from plant raw Materials, critical roles are played not only by the control of starting materials, storage, and Processing conditions, but also by their origin (Background history). Important Factors influencing the quality of herbal raw materials—and consequently determining their subsequent quality and therapeutic efficacy—include agrophytological indicators (background agricultural practices), Selection of cultivars, and conditions of cultivation, harvesting, and drying. These factors can significantly affect the microbial bioburden of plants.

Class="center">Fig. 30. Microbial contamination of plants.

The constancy and stability of plant microflora are likewise governed by external factors stemming from natural, agricultural, environmental, and technological influences (Table 11).

Table 11. Factors Determining the microbiological quality of plants.

Microorganisms are permanent companions not only of humans and animals, but equally of higher plants, including those used as medicinal raw materials. More than 200 species of medicinal plants are utilized in Russia. Microorganisms colonize and thrive both On the surface and within the green Tissues, roots, seeds, and fruits of plants. Microbiological data on medicinal plants have been compiled and presented in Table 12 (total count of aerobic mesophilic bacteria) and Table 13 (enterobacteria).

Table 12. Species composition of mycoflora isolated from medicinal plant materials and raw products

Table 13. Microbiological purity of herbal medicinal preparations

These tables list A large number of plants along with their respective microbial count ranges. It is quite apparent that the distinct variations in total aerobic bacterial counts (Table 13) reflect the original and ecological criteria described above. For example, the relatively low bacterial growth found in certain plants (such as bilberry fruits, Myrtilli fructus) may be attributed to natural antibacterial substances, whereas a high microbial count (e.g., Herba urticae) may indicate less favorable hygienic conditions. Furthermore, factors such as the distance of the plant from the soil and The ratio of plant surface area to sample weight can play a definite role. Although enterobacteria are ubiquitous in nature, the presence of members of this family indicates fecal contamination. Together with coliform bacteria, they can serve as an indicator of poor hygienic conditions. Reports regarding the presence of pathogenic microorganisms on medicinal plants are relatively limited, yet contamination by pathogens cannot be entirely ruled out. Recently, Czech et al. [15] tested for a wide range of pathogens as indicator microorganisms. They demonstrated that these microorganisms are detected relatively infrequently, with the exceptions of Bacillus cereus and Clostridium perfringens. In the 1980s, Leimbeck [14] detected E. coli and Pseudomonas aeruginosa in numerous samples and therefore recommended treating herbal remedies with boiling water to decontaminate them from microorganisms. Currently, it has been shown that plant-microbial interactions involve Plant Growth-Promoting Bacteria (PGPB). These include, for instance, certain strains of the genus Pseudomonas that protect plants against frost by preventing ice crystal formation on the aerial PARTS OF THE plant during brief periods of sharp temperature drops. Representatives of the genera Bacillus, Agrobacterium, and Pseudomonas act as sources of biocontrol agents [14].

Bacteria cause over 200 plant diseases. Bacterial infections inflict severe damage on vegetable, fruit, and industrial crops. The length of a bacterial Cell ranges from 0.5 to 4.1 µm, with a diameter of 0.3 to 0.8 µm. The predominant shape of Gram-negative phytopathogenic bacteria is rod-like (with the exception of Streptomyces, which have a filamentous structure). Most phytopathogenic bacteria are motile via flagella [with only a few non-motile forms]. In most motile phytopathogenic bacteria, the flagella are polar, while peritrichous arrangement is less common.

Given that widely used medicinal plants harbor a significant amount of mycotoxin-producing Molds, products obtained by cold water maceration must be monitored with particular care. Hitokoto et al. demonstrated that genera such as Penicillium, Aspergillus, Rhizopus, Mucor, Cladosporium, and Aureobasidium spp. are frequently associated with herbal medicinal preparations, whereas mycotoxin producers accounted for only 2% of these isolates. Conversely, Kumar and Roy [16] detected high levels of aflatoxin risk in several samples of medicinal plants belonging to various taxa. In light of these findings, it is evident that environmental conditions (climate, humidity, hygiene, etc.) heavily contribute to the presence of mycotoxins.

Although bacterial endospores and fungal spores can be regarded as the two dominant groups of contaminants associated with medicinal plants, A wide variety of bacterial Cells, fungal cells, and viruses can reside on and within the plant. Pathogenic microorganisms may also be present among them, a fact that severely restricts The Use of these plants beyond simply reducing quality due to spoilage. Moreover, by analogy with culinary spices and herbs, the presence of contaminants from rodents, insects, and inorganic sources (e.g., stones) cannot be ruled out in certain preparations.

Certain plants (such as calamus, lemon balm, thyme, basil, sweet fennel, etc.) contain natural barriers and antibacterial substances that exert typical inhibitory effects on Microbial growth AND stability. It is estimated that approximately 1,400 herbs and spices may contain antibacterial agents of diverse chemical nature, such as Essential Oils, Peptides, and liquid or organic extracts.

Some of these biological activities have been evaluated for specific therapeutic uses and integrated into clinical practice. Additionally, it is quite expected that certain antioxidants naturally present in plants may restrict the growth of certain microbes.

Mycorrhiza is the most widespread type of plant-microbe Symbiosis (PMS), established between plants and fungi that colonize roots and other subterranean Organs, wherein a part of the mycobiont (the fungal partner) resides inside the plant while the other part remains in the soil. The compartmentalization of the mycobiont into intraradical and extraradical regions reflects its key role as a mediator between the plant and the soil. In natural phytocenoses, where intense competition for soil nutrients occurs, independent plant survival is severely hindered, making the dependence of most plants on mycorrhizal fungi ecologically obligate. This function holds global ecological significance, as only a few plants (such as ephemerals and aquatic forms) can independently meet their mineral Nutrition and water requirements. Under artificial supply of complete mineral nutrients (e.g., in hydroponic systems), most plants are capable of completing their life cycle without mycorrhizae. In orchids (Orchidaceae), this dependency is even profound, as they cannot undergo normal development without the fungi required for seed germination, Embryogenesis, and the nourishment of chlorophyll-free seedlings (Finlay, 2008).

Being multicellular eukaryotes, mycorrhizal [14] fungi undergo a symbiosis-specific morphogenesis, forming structures that do not develop in the absence of a host interaction. Based on the Organization OF THE intraradical portion of the mycobiont, mycorrhizal symbioses are divided into endomycorrhizae (where the fungus penetrates PLANT CELLS AND forms specialized structures within them that ensure the closest possible cellular contact between the partners) and ectomycorrhizae (where the fungal spread is restricted to the intercellular space). The universal form of endomycorrhiza is arbuscular mycorrhiza (AM), formed by the overwhelming majority (80-90% of species) of terrestrial plants. Ectomycorrhizae (EM) are restricted to woody and shrubby angiosperms.

The host plant's contribution to The formation of mycorrhizal structures is not as substantial as its contribution to root nodules: it is limited to organizing close cellular contacts with the mycobiont (AM) or modifying root architecture (EM). The most pronounced symbiotic development is observed in orchid roots, which may be heavily reduced or reorganized into structures devoid of assimilatory function and specialized for supporting and nourishing the mycobiont.

Microorganisms that normally develop on The surface of plants are classified as epiphytes (from Greek epi — upon, phyton — plant). They are harmless, act as antagonists to certain phytopathogenic microorganisms, and grow at the expense of ordinary plant exudates and organic contaminants on the plant surface. Epiphytic microflora prevents the penetration of phytopathogenic microorganisms into plant tissues, thereby enhancing plant Immunity. The bulk of the epiphytic microflora consists of Gram-negative bacteria *Erwinia herbicola*, which form golden-yellow colonies on meat-peptone Agar. These bacteria act as antagonists to the soft rot pathogen of vegetables. Other bacteria are also normally detected, such as *Pseudomonas fluorescens*, less frequently *Bacillus mesentericus*, and small quantities of fungi.

Diseases caused by bacteria are referred to as bacterioses. Pathogens causing bacterioses include pseudomonads, mycobacteria, erwinias, corynebacteria, agrobacteria, etc. Bacterioses encompass various types of rots, tissue necroses, plant wilting, tumor development, etc. A distinction is made between generalized and localized bacterioses. Generalized bacterioses lead to the death of the entire plant or its individual parts. They may manifest on the roots (root rots) or within the plant Vascular System. Localized bacterioses are restricted to the infection of specific plant parts, manifesting in parenchymal tissues. The genus *Erwinia* includes species that cause blight, wilt, and wet or watery rot diseases, for example, *E. amylovora*—the CAUSATIVE AGENT OF fire blight in apples and pears, and *E. carotovora*—the causative agent of bacterial soft rot. The genus *Pseudomonas* comprises various species, notably those causing bacterial leaf spot (*P. syringae*, etc.), where spots of varying color and size develop on the leaves depending on the plant species.

3.1 Microbial-Plant Interactions in Plant GROWTH AND DEVELOPMENT

As a logical starting point for studying microorganism-plant interactions, we can look at seed germination in soil. However, plant seeds that enter the soil are already colonized by microorganisms, meaning that microbial-plant relationships begin much earlier. Quite often—and this is particularly typical of phytopathogens—microorganisms are already present inside the mature seed. Potentially, a plant seed may carry bacterial cells, their endospores or cysts, conidiospores and/or actinomycete hyphal fragments, fungal mycelial fragments and/or their conidiospores, protozoan cysts, and possibly nematode eggs and viruses. The Abundance of various microorganism groups varies and depends on many factors, including the size, shape, taxonomic genus and species of the plant, the presence or absence of specific seed coats, and the plant's habitat (i.e., geographical and climatic factors). The abundance and diversity of microorganisms colonizing the seed surface are also largely determined by the biology of the microorganisms themselves. Such characteristics as size and Morphology, cell surface structure, and The ability to survive for prolonged periods under low-humidity conditions (desiccation), exposure to light, and so forth play a crucial role in seed surface contamination and retention. Consequently, it is virtually impossible to predict the exact number of bacteria, let alone fungi, present on the surface of a healthy seed.

Bacteria belonging to the genera Agrobacterium, Arthrobacter, Bacillus, Burkholderia, Clavibacter, Clostridium, Curtobacterium, Erwinia, Pseudomonas, Rhizobacter, Rhizomonas, Streptomyces, Xanthomonas, and others, as well as fungi of the genera Acremonium, Alternaria, Aureobasidium, Aspergillus, Botrytis, Cephalosporidium, Claviceps, Drechslera, Fusarium, Gibberella, Helminthosporium, Humicola, Penicillium, Peronospora, Phoma, Phytophthora, Plasmopara, Puccinia, Pythium, Rhizoctonia, Septoria, Trichothecium, Ustilago, Verticillium, and others, can be found on the surface, within the seed coats, and in some cases even in the tissues of various seeds. Among the listed genera of bacteria and fungi, many are true phytopathogens.

Metabolic processes occur within plant seeds, even when they are in a state of deep dormancy, and consequently, exchange processes with the environment take place. Many plant seeds possess a specific, species-characteristic odor due to the synthesis of volatile Organic compounds. These substances serve as potential substrates for microorganisms. Naturally, microorganisms inhabiting the surfaces of seeds and plants constantly experience the effects of these metabolic processes to a greater or lesser degree.

Upon encountering favorable moisture and temperature conditions, a plant seed swells and germinates (Fig. 31). During Swelling—and especially during germination—corresponding molecular-genetic and physiological-biochemical processes take place within the seed. These same factors, moisture and temperature, exert a corresponding effect on the microorganisms located on the seed surface. However, the primary impact on the seed surface microbial community is driven by the "release" of organic substances from the swelling and germinating seed. The concentration and composition of such substances are species-specific. For instance, the germination of wheat seeds reveals CARBOHYDRATES (mainly glucose and fructose, comprising up to 10 components in total), organic acids (mostly succinate, fumarate, and malate), and up to 16 Amino Acids, among which aspartic and glutamic acids dominate.

Fig. 31. A plant germinating and developing from a seed in the soil [21].

In addition to non-volatile substances, the germination of seeds and the activation of surface microbial METABOLISM are accompanied by the release of volatile organic compounds (VOCs). The mutual influence of these substances on macro- and microorganisms is currently an area of active research.

A plant germinating and developing from a seed in the soil (Fig. 31) encounters various microscopic biological entities: microscopic animals, Protozoa, fungi, bacteria, and viruses. The future plant interacts with these entities via both its developing root System and Its future aerial part—the stem, while still a seedling. The root comes into contact with non-specific microorganisms (i.e., those whose contact does not lead to infection) and specific, root-infecting microorganisms. Among the infectious agents are non-pathogenic forms and typical pathogens. Non-pathogenic forms include, for example, root-nodule bacteria, and among fungi—mycorrhizal fungi, both endo- and ectomycorrhizal. However, another viewpoint exists, in which the interaction of these bacteria and fungi with plants is viewed in a broader context as Pathogenesis. Among the structural parts of the soil,

organic matter—humus, consisting of the remains of animal and plant organisms and soil-dwelling microbes—is of particular interest to microbiology. The surface layer of the soil is poorer in microbes because they are adversely affected by environmental factors such as drying, ultraviolet rays, sunlight, elevated temperatures, and others. The highest concentration of microorganisms is found at a depth of 5–15 cm, decreasing at 20–30 cm, and dropping even further at 30–40 cm. Cultivated soils are the richest in microflora, whereas sandy, mountainous, and vegetation-free soils are the poorest; the microbial content in soils increases from north to south.

Typical soil bacteria include Bacillus subtilis, Bacillus mycoides, Bacillus mesentericus, Bacillus megatherium, Clostridium tetani, Clostridium perfringens, Clostridium oedomaticus, Clostridium histolyticus, Clostridium botulinum, Clostridium chauvoei, as well as thermophilic, pigmented, non-pigmented, and other microorganisms that sometimes account for 80–90% of the total soil microflora. The space and soil surrounding the developing root are referred to as the rhizosphere. The first Definition of the rhizosphere is generally attributed to Hiltner in 1904. Today, the rhizosphere is understood to be the region surrounding the root from 0 to 2–8 mm in diameter, where microorganisms proliferate more abundantly due to The stimulation of their growth by root exudates or, more broadly, root deposits. The rhizosphere region is sometimes also called the endorhizosphere, a concept that includes the root tissues themselves, in contrast to the rhizoplane, which refers strictly to what is located directly on and attached to the root surface. Root exudates are low-molecular-weight organic substances, products of plant Photosynthesis and metabolism. They include sugars, organic acids, amino acids, alcohols, Hormones, Vitamins, and others. These substances "leak" from the zone near the root tip, specifically the root "elongation" zone during its growth and development. Root rhizodeposits encompass a broader concept. They include not only exudates but all other substances as well: high-polymer mucilages of polysaccharide and protein nature, Enzymes, dying and sloughing surface cells with their contents, tissue fragments (in particular, the cortex of upper Aging root sections), the root cap, root hairs, volatile organic compounds, and others. It is estimated that a plant loses more than 30–40% of its photosynthetic products in the form of rhizodeposits.

In addition to the chemical impact on the soil and the microorganisms within it—including changes in pH and Eh—the growing root also exerts a purely mechanical effect on its surrounding econiche. The phenomenon of higher microbial density around the root resulting from the consumption of exudates and rhizodeposits is known as the rhizosphere effect. Comparative experiments involving sterile plants grown in sterile and non-sterile soils have demonstrated that microbial-plant interactions in the rhizosphere are manifested, among other ways, in the stimulation of plant exudation. The abundance of Microorganisms in the rhizosphere can exceed that in the surrounding soil by several percent to tens of percent, or even by an order of magnitude.

Determining the total number of microorganisms in the rhizosphere is extremely difficult. It depends on soil type, plant species, and other factors, and can range from millions to hundreds of billions of cells per gram of dry soil.

It has been found that the spatiotemporal organization of the rhizosphere microbial community does not simply reflect the root exudation zones, but possesses its own specific structure. The microbial community develops in a wave-like pattern along the growing root, meaning that zones of higher microbial density alternate with zones of low density. Furthermore, peaks of varying microbial density shift over time along the root, allowing us to speak of a "moving wave" form of rhizosphere microbial community development. Wave-like development of microbial populations has been observed in both the rhizoplane and the direction perpendicular to the root, indicating that the phenomenon of wave-like microbial growth is a universal pattern of rhizosphere microbial community development.

The space surrounding the aerial surface of a plant, including the plant's tissues, is called the phyllosphere, and the plant surface itself is the phylloplane. Microorganisms colonizing the aboveground plant surfaces are referred to as epiphytic microorganisms (from the Greek epi—around, phytos—plant). The number of epiphytic microorganisms found on leaf surfaces can sometimes reach 10⁸ cells per gram of fresh leaves, or 10⁶ per 1 cm², which is quite comparable to the microbial population in a gram of soil. The abundance and diversity of microorganisms, such as bacteria, depend heavily on the plant species, its habitat, climate, weather conditions, and several other factors. Some of these have already been mentioned in the Discussion of microorganisms on plant seeds. These are again saprotrophic and phytopathogenic representatives of the genera Pseudomonas (P. syringae, P. fluorescens, etc.), Erwinia (E. carotovora, E. amylovora), Xanthomonas (X. campestris), Agrobacterium (A. tumefaciens), as well as Beijerinckia, Enterobacter, Klebsiella, Methylobacterium, and many others. There are differences between the microbial communities on the upper and lower sides of a leaf, with light and temperature playing a significant role. Naturally, desert plants and succulents harbor far fewer microorganisms per unit surface area or per gram than plants in tropical rain forests.

As mentioned above, a large number of rhizosphere microorganisms reside in the soil near plant roots. These include pseudomonads, mycobacteria (non-spore-forming rods), actinomycetes, as well as spore-forming bacteria and fungi. These microorganisms convert various complex substances into compounds accessible to plants, synthesize biologically active compounds (vitamins, Antibiotics, etc.), enter into symbiotic relationships with plants, and exhibit antagonistic properties against phytopathogenic microbes. The composition of the rhizosphere is specific to each plant species. Plants in cultivated soils are more heavily contaminated with microorganisms than those in meadows and forests. Microbes are particularly abundant in the lower parts of plants due to their transfer from the soil. Large numbers of microorganisms are found on plants growing in garbage dumps, near manure, and in livestock grazing areas. Pathogenic microorganisms may also be detected on such plants.

The microbial contamination of herbal medicinal raw materials depends on the initial level of bioburden, but it can increase during the stages of primary processing, grinding, and standardization. Spoilage of raw materials occurs mainly under conditions of elevated humidity, which promotes the proliferation of putrefactive microorganisms. Microorganisms are most abundant in the soil, particularly in the root zone. This zone contains various mycobacteria, pseudomonads, spore-forming, nitrogen-fixing, and nitrifying bacteria, actinomycetes, and fungi. The region around plant roots is a zone of intensive growth and heightened microbial activity. The surface of The Root System is colonized predominantly by pseudomonads and fungi. The latter enter into symbiotic relationships with plants and form mycorrhiza (fungus-root), which stimulates the growth of both partners.

3.2 Phytopathogenic Fungi

Phytopathogenic microorganisms include bacteria, viruses, and fungi. Phytopathogenic fungi cause mycophytoses [20].

Fungi causing diseases in forest trees and cultivated plants inflict enormous economic damage. Mass plant diseases (epiphytotics) can cause famine in entire populations across countries. Diseases reduce potential crop yields by 10–20% or more. Central Europe accounts for up to 162 serious crop diseases, of which 135 (83%) are caused by fungi, with the remainder caused by bacteria and viruses. By infecting medicinal plants, fungi—like other microorganisms—render them unsuitable for use as raw materials in the pharmaceutical industry (descriptions of phytopathogenic fungi and bacteria are provided in Chapter 3).

Parasitic fungi are subdivided into ectoparasites and endoparasites. Ectoparasites spread primarily across leaves, penetrating host cells only through specialized organs (such as haustoria) and sporulating on the plant surface. These include the causative agents of powdery mildew in cereals, cucurbits, tobacco, and potatoes. Endoparasites develop their thallus inside the host and emerge to the surface during sporulation.

Depending on the type of relationship between the fungus and the host plant, parasites are distinguished into obligate and facultative. The former infect only living tissues and fail to grow on standard nutrient media. Such fungi include economically vital phytopathogenic fungi, for example, Puccinia graminis (cereal stem rust) and Peronospora tabacina (tobacco downy mildew).

Facultative parasites also infect living plant tissues, but they do not die off when the host dies; they are capable of growing on laboratory nutrient media. These include numerous wood-decaying fungi, which are of practical interest in connection with agricultural and forestry waste management.

Phytopathogenic fungi vary in their host Specificity. For instance, among powdery mildew pathogens of the order Erisiphales, there are both species that infect only specific host races and fungi that attack plants from different orders. The rust fungus Puccinia graminis develops its haploid phase on barberry and its dikaryotic phase on cereals. Some parasitic fungi show a preference for specific plant organs. For example, the ergot fungus Claveliniceps purpurea infects only cereal Ovaries, and Ustilago violacea (the smuts pathogen) infects only stamens. Fungi of the genus Verticillium develop within the plant's vascular system, disrupting sap flow and leading to wilting and death.

The fungus penetrates plant tissue through Stomata or surface wounds; they are capable of breaching surface structures with their infection hyphae. Subsequently, the fungus spreads throughout the plant, accompanied by the appearance of disease symptoms. At the next stage of the infectious process, the fungus develops sporulation structures, while the plant's response depends on the quantity and quality of the pathogen.

3.3 Phytopathogenic Bacteria (Plant Pathogens)

Various viruses, bacteria, and fungi (phytopathogenic microflora) are capable of causing plant diseases [15, 20]. Plant Infections caused by

phytopathogenic bacteria are known as bacterioses. Depending on the localization of the pathological process, they are classified into general and local infections. The former lead to the death of the entire plant or its individual parts, while the latter affect specific plant areas. Based on The Mechanism of damage, bacterioses are subdivided into parenchymatous diseases, vascular infections, and tumors.

Parenchymatous diseases develop when bacteria invade plant tissues through various anatomical openings (stomata, lenticels, nectaries) and integumentary tissue injuries. The pathogens secrete enzymes and toxins that facilitate their spread through intercellular spaces. The penetration of bacteria deep into the tissue causes mass cell death. These diseases include rots (primarily caused by bacteria of the genera Pseudomonas and Erwinia), blights (mainly caused by Erwinia and Corynebacterium species), and leaf spots (principally caused by Pseudomonas and Xanthomonas species).

Vascular infections develop when bacteria spread through the plant's vascular system. The primary causative agents are Corynebacterium species (C. fascians, C. insidiosum, C. michiganense). The bacteria multiply within the vessels, causing clogging due to damage to the vessel walls, which ultimately leads to plant wilting.

Microbes inhabiting medicinal plant materials may comprise members of the normal epiphytic and phytopathogenic microflora. The composition of the normal plant microflora depends on the plant species, age, soil type, ambient temperature and humidity, other growing conditions, as well as the timing of inspection, plant height, and integrity. Normally, microorganisms (epiphytic microflora) inhabit the surface of living plants (leaves, stems, fruits, seeds). They feed on plant secretions and organic pollutants present on the plant surface. Epiphytic microflora is non-pathogenic and prevents phytopathogenic microbes from penetrating plant tissues (exhibiting an antagonistic effect). The epiphytic microflora includes: Gram-negative (G^-) bacteria Erwinia herbicola (forming golden-yellow colonies on meat-peptone agar [MPA]), the Gram-negative polymorphic rod Pseudomonas fluorescens (forming transparent fluorescent colonies on MPA). These bacteria produce the green pigment pyoverdine, which causes colonies to fluoresce under short-wave UV radiation. Pyoverdine possesses bacteriocin properties, acting against both Gram-positive and Gram-negative bacteria, and also exhibits moderate fungicidal activity. The epiphytic microflora also includes Gram-positive (G⁺) spore-forming bacteria such as Bacillus mesentericus and Bacillus vulgatus, as well as non-spore-forming bacteria like Bacterium putidam and E. coli, alongside a small number of fungi. In autumn, a higher microbe count is found on leaves than in early spring. An increase in air humidity leads to a higher microbial population on plant surfaces (epiphytic microflora). Damaged plants accumulate large amounts of dust and, consequently, a higher concentration of microorganisms.

The surface of some phytopathogenic bacteria is covered with a slimy layer—a capsule. This capsule plays a crucial role in bacterial survival under adverse conditions, rendering them resistant to sunlight, chemical agents, and other environmental factors. In humid weather, bacterial cells accumulate on the surface of infected organs as an exudate (mucous mass). Regarding their nutrition, phytopathogenic bacteria are heterotrophs capable of growing on nutrient media. On solid nutrient media, bacteria form colonies whose pigmentation, shape, and surface characteristics are typical of the specific strain.

Phytopathogenic bacteria belong to the same genera and species as saprotrophic forms.

Disease Symptoms and Types of Bacterioses

Depending on The impact of bacteria on the plant and the extent of tissue damage, bacterioses are divided into two main types: diffuse (or systemic) and local.

In diffuse bacterioses, the pathogen invades The vascular system, spreads through the

conducting bundles and adjacent tissues. This disrupts water uptake, causing the plant to wilt. Wilting is the primary symptom of systemic bacterioses. Symptoms of certain bacterioses are illustrated in Fig. 32. Wilting refers to pathological Changes in the tissues of individual organs or the entire plant associated with the loss of turgor. As the bacteria multiply, they first clog the vascular bundles of individual organs and subsequently those of the entire plant, leading to systemic wilting. For instance, bacterial wilt of tomato first manifests only in individual leaves, later on certain shoots, and finally results in the complete wilting of the plant under the action of the pathogen Clavibacter michiganensis subsp. michiganensis.

Fig. 32. Diffuse bacteriosis. Potato ring rot: a) infected plant, b) infected tuber.

Bacterial plant diseases are phytopathoses caused by bacteria of the genera Pseudomonas and Erwinia. They cause severe damage to numerous plant species. Infections can be general, leading to the death of the entire plant or its parts, manifesting in roots (root rots) or the vascular system (Vascular Diseases); local, restricted to specific parts or Organs of the plant, as well as affecting parenchymal tissues (parenchymatous diseases such as rots, leaf spots, and blights); or they may be of a mixed nature. A special category consists of bacterioses associated with the formation of new growths (tumors).

Causative agents of bacterioses are predominantly non-spore-forming bacteria belonging to the families Mycobacteriaceae, Pseudomonadaceae, and Bacteriaceae. Among them, there are polyphagous bacteria that affect a wide range of plant species, as well as specialized forms that target closely related plants of the same species or genus. Polyphagous bacteria cause the most common bacterioses, such as soft rots and crown gall in various fruit trees and grapevines.

Specialized bacteria cause bacterial leaf spot of bean, cucumber bacteriosis, black bacterial spot and bacterial canker of tomato, vascular bacteriosis of cabbage, tobacco wildfire, black chaff and basal bacteriosis of wheat, bacterial blight of stone fruits, pears, mulberry, and citrus crops, ring rot and blackleg of potato, cotton gummosis, striped blight of millet and barley, among other diseases.

The onset and development of bacteriosis [17] depend on the presence of the infectious agent and a susceptible host plant, as well as environmental factors. By modifying these factors, the course of the infection process can be managed. For example, cucumber bacteriosis in greenhouses develops only in the presence of free water droplets (liquid moisture) and air temperatures of 19–24°C. Ventilating greenhouses and raising the air temperature help arrest disease progression. As noted previously, bacteria enter plants through various wounds and natural openings; for instance, pathogens causing various leaf spots penetrate via stomata, those causing fruit tree blight through floral nectaries, and vascular bacterioses of crucifers through hydathodes (water pores) in the leaves. In addition to high air humidity and temperature, disease development is promoted by water droplets on plants, a deficiency of phosphorus and potassium, and high soil pH.

3.4 Bacterial Spot, Bacterial Blight, and Vascular Bacteriosis

The disease predominantly affects young leaves and shoots. Depending on the pathogen species, bacterial spots present with various symptoms. The most characteristic clinical picture begins with the formation of small, water-soaked spots on the leaf or stem surface, which gradually turn black. Typically, these spots are irregularly angular in shape and surrounded by a yellow or light-green halo. The bacteria generally spread along the Veins. The spots enlarge, coalesce, and ultimately cause the entire leaf to blacken, eventually resulting in plant death.

Optimal conditions for bacterial proliferation are temperatures of 25–30°C and high air humidity. Bacterial mortality occurs only at temperatures above 56°C. Bacteria of the genus Xanthomonas are resistant to desiccation and can endure low temperatures for extended periods.

A variant of bacterial spot is the so-called bacterial blight caused by bacteria of the genus Pseudomonas. In this case, plants develop fairly large, shapeless blackened areas rather than distinct spots, which subsequently dry out, giving the affected leaf area a scorched appearance. Under favorable environmental conditions, the disease progresses very rapidly, causing necrosis of individual parts and the death of the entire plant. Bacterial blight typically initiates on young leaves, shoots, and flowers. The bacteria penetrate the plant via stomata or minor wounds and begin to multiply in the intercellular spaces of the leaf parenchyma. The incubation period of the disease ranges from 3 to 6 days, depending on temperature. The bacteria overwinter and persist in the soil and on seeds.

Source of Infection

Seeds are one of the most critical sources of infection. During germination, infected seeds can transmit the pathogen to seedlings, which then migrate through the vascular system to infect mature plants during the growing season. Furthermore, diseased seeds can serve as a vehicle for spreading the infection, triggering bacterioses in regions where they were previously absent. The infection can also be disseminated by healthy-looking green plants that harbor the bacteria, allowing pathogens to be transported to new areas of the country via infected plant material (cuttings, budwood/buds). Plant debris constitutes another primary source of infection. Phytopathogenic bacteria survive particularly long and well within woody plant tissues.

Soil as a source of infection does not pose a major threat. Numerous studies have demonstrated that phytopathogenic microbes introduced into the soil rapidly perish due to the action of antagonistic microorganisms (a process akin to soil self-purification).

Some insect species can also serve as a source of primary infection. Raindrops containing small particles of infected plant residues pose a major threat in spreading bacterioses, as wind and air currents carry them over long distances (air itself does not play a direct role in disease transmission). Water—such as irrigation water, river water, and other sources—can also transmit phytopathogenic bacteria. Finally, nematodes play a significant role in the natural spread of bacterioses.

Local bacterioses manifest as damage to the parenchymal tissue of individual plant organs. Their main symptoms include necroses, chloroses, rots, and tumors. A local type of rot is observed, for example, when apricot fruits are infected (fruit browning). The rot localizes near the fruit pit; the disease is caused by the soil-dwelling, spore-forming bacterium Bacillus mesentericus (Fig. 33).

Fig. 33. Local bacteriosis.

Some bacterioses are characterized by the appearance of bacterial exudate. It is secreted by the causative agents of fire blight of pome fruits (Erwinia amylovora), angular leaf spot of cucumber (Ps. syringae pv. lachrymans), common blight of bean (X. phaseoli), and other bacteria, predominantly under high air humidity.

Control and Prevention Measures [16]

Some diseases are seed-borne, making seed disinfection an essential measure. Crop residues left on the soil surface serve as one of the sources of infection. Therefore, deep moldboard plowing is effective, as it buries pathogens deep into the soil where they perish due to the antagonistic activity of bacteria and actinomycetes or are consumed by protozoa. Collecting and destroying fallen leaves, fruits, and branches, along with proper crop rotation to prevent the accumulation of pathogens in the soil, are of great importance.

Chemical plant protection measures are widely utilized. Proper soil cultivation and fertilizer application are highly significant as they enhance the resistance of cultivated crops. Breeding and selecting disease-resistant varieties are crucial, and hypersensitive varieties are also of great interest, as their rapid cell death halts The Development of the parasitic fungus. To prevent The transport of pathogens from one country to another, special quarantine services carry out dedicated phytosanitary measures.

A plant can be saved if the bacteriosis has not yet affected the entire vascular system or is localized in nature (e.g., rot starting from the leaf tip). If the roots have rotted, one can still attempt to root the top cutting (provided the plant is capable of propagation via cuttings). If the rot has affected only part of the roots while the above-ground portion appears healthy, an attempt can be made to save the plant: free the roots from soil, trim away all rotted parts, transplant into freshly prepared dry soil, water, and spray with Bordeaux mixture (or other copper-based treatments). The infection will not spread to a neighboring plant, but all tools and pots must be thoroughly disinfected.

During germination, seeds can infect seedlings, and subsequently travel through the vascular system to infect mature plants during the growing season. Furthermore, diseased seeds can act as a source of infection spread, causing bacterioses to appear in areas where they were previously absent. Green plants can also disseminate the infection, as bacteria survive well within them and are transported to new Regions of the country alongside infected plant material (cuttings, budwood—buds). Diseased plant residues represent one of the primary sources of infection by bacterioses. Phytopathogenic bacteria survive particularly well and for long periods in the woody parts of plants.

Some insect species can also serve as a source of primary infection. Raindrops containing small particles of infected plant residues pose a major threat in spreading bacterioses, as wind and air currents carry them over long distances (air itself does not play a direct role in disease transmission). Water—such as irrigation water, river water, and other sources—can also transmit phytopathogenic bacteria. Finally, nematodes play a significant role in the natural spread of bacterioses.

3.5 Mycoplasmas (Phytoplasmas)

Phytoplasmas are classified as prokaryotes, meaning organisms that lack a true Nucleus. The nuclear apparatus in these organisms is commonly referred to as the nucleoid.

Mycoplasmas have long been known as causative agents of Human and Animal diseases. Plant-pathogenic mycoplasmas (phytoplasmas) were discovered only in 1967. They were detected by Japanese scientists using an Electron microscope in the phloem of mulberry plants affected by dwarfism. These mycoplasma-like organisms (MLOs) proved to be phytopathogenic. It was established that they are transmitted from plant to plant by leafhoppers, psyllids (xylids), and dodder, causing diseases resembling "witches' brooms" and yellows (Fig. 34). In their properties, MLOs resembled organisms belonging to the mycoplasma group. However, unlike animal mycoplasmas, which are typically found extracellularly, phytoplasmas were detected intracellularly.

Fig. 34. Symptoms of phytoplasmoses: a — tomato stolbur; b — potato "witches' brooms"; c — clover flower virescence (phyllody).

Phytoplasmas are a specific group of phytopathogenic organisms occupying an intermediate position between bacteria and viruses. Their cells are generally spherical, though some have an elongated or dumbbell shape. Cell diameter ranges from 0.1 to 1 µm.

Phytoplasmas lack a true Cell wall and therefore do not have a fixed shape; they are surrounded by a trilaminar elementary membrane, which distinguishes them from bacteria.

Compared to viruses, they are characterized by cellular structure and the ability to reproduce on artificial nutrient media. On solid media, they form small, specific colonies visually resembling a "fried egg." Unlike Viral Particles, phytoplasma cells contain two Types of Nucleic acids (DNA and RNA) and Ribosomes similar in size to bacterial ribosomes. Unlike bacteria, phytoplasmas are resistant to penicillin, yet sensitive to tetracycline compared to viruses.

The primary vectors of phytoplasmas are leafhoppers, psyllids, and planthoppers. Phytoplasmas can survive only in living plant tissues: tubers, rhizomes, bulbs, and the rootstocks of perennial weeds. Within vector insects, phytoplasmas can persist and multiply for long periods.

Rickettsiae. In 1972, organisms morphologically similar to rickettsiae—Obligate Intracellular Parasites of vertebrate and invertebrate animals—were discovered in the phloem of clover plants with deformed leaves, and were named rickettsia-like organisms.

The primary agents of plant tumors are bacteria of the genus Agrobacterium (most commonly A. tumefaciens). Agroviruses contain oncogenic Plasmids. Following their transfer into plant cells, specific tumors—crown galls—develop. The causative agents of mykophytoses also induce parenchymal and vascular damage in plants. The use of raw materials contaminated with fungi as food products can cause severe diseases known as mycotoxicoses. Phytopathogenic viruses cause mosaic diseases, yellows, and stunting. Their characteristic feature is the appearance of pale spots or entire discolored areas, as well as delayed plant growth. In addition to viruses, Viroids are also classified as phytopathogens.

Almost all phytopathogenic bacteria are Gram-negative; only species of the genera Clavibacter and Streptomyces yield a positive reaction.