Forest Microbiology: Volume 2: Forest Tree Health

Forest Microbiology, Volume Two: Forest Tree Health highlights a range of emerging microbial phytopathogens of forest trees, along with novel approaches for managing tree pests and diseases in a changing climate. The book provides an overview of selected microbial pathogens of forest trees, with an emphasis on their biology, lifecycle, spreading mechanisms, impact on affected tree species and current and prospective control strategies. At the same time, the impact of tree microbiomes on host fitness is discussed. Beneficial components of tree microbiota are presented, along with their functional role in tree nutrition, immunity and disease resistance.

In addition, this volume addresses the many functions of microbial disease agents of trees including fungi, bacteria, viruses and phytoplasma. Strong emphasis is placed on the genetics, biochemistry, physiology, evolutionary biology and population dynamics of the microorganisms involved. This title is a key resource for foresters and forest pathology practitioners, as well as plant biologists.

• Provides an overview of selected microbial pathogens of forest trees, with an emphasis on their biology, lifecycle, spreading mechanisms, impact on affected tree species and current and prospective control strategies
• Highlights novel approaches to managing tree pests and diseases in a changing climate
• Addresses the many functions of microbial disease agents of trees, including fungi, fungi, bacteria, viruses and phytoplasma

• Language: English
• Publisher: Academic Press
• Release date: Jul 1, 2022
• ISBN: 9780323984485

Book preview

Part I

Basis of tree pathology

Chapter 1: Basic concepts and principles of forest pathology

Fred O. Asiegbu    Faculty of Agriculture and Forestry, University of Helsinki, Helsinki, Finland

Abstract

Forest pathology is the study of diseases of woody plants growing in natural forests, plantations, tree nurseries, and urban environments. As a science, it is dedicated to understanding the nature of tree diseases caused by pathogens and their insect vectors as well as the ecology and biology of the pathogenic organisms and infectious agents (fungi, oomycetes, bacteria, phytoplasma, viruses, nematodes, protozoa) of forest trees. It is a discipline within forestry that serves the public and environmental interest by applying scientific principles to the prevention and control of tree diseases. Both biotic and abiotic factors play significant roles in forest tree diseases. Of the biotic factors, fungal diseases are primarily the most widely studied. Among abiotic factors, diseases and disorders caused by air pollution and by climatic and edaphic factors are of major importance. This chapter also discusses the concept of disease in tree pathology as well as nature and symptoms of tree diseases. The diverse lifestyles and nutritional strategies of tree pathogens were highlighted. Additionally, the basic principles of wood decay and wound healing in living trees as well as important components of disease resistance strategy of forest trees against phytopathogens were equally addressed.

Keywords

Tree pathology; Disease triangle; Disease cycle; Disease resistance; Wood decay; Wound healing

1: Definition of forest tree pathology

Forest pathology is the study of diseases of woody plants growing in natural forests, plantations, tree nurseries, and urban environments. As a science, it is dedicated to understanding the nature of tree diseases caused by pathogens and their insect vectors as well as the ecology and biology of the pathogenic organisms and infectious agents (fungi, oomycetes, bacteria, phytoplasma, viruses, nematodes, protozoa) of forest trees. It is a discipline within forestry that serves the public and environmental interest by applying scientific principles to the prevention and control of tree diseases. Both biotic and abiotic factors play significant roles in forest tree diseases. Of the biotic factors, fungal diseases are primarily the most widely studied. Among abiotic factors, diseases and disorders caused by air pollution and by climatic and edaphic factors are of major importance.

2: Historical background of forest pathology

On a broader perspective, disease refers to a condition that negatively disrupts the normal physiological functioning or causes disorder of structure or part of an organism including forest trees. Biological agent capable of negatively disrupting the normal metabolic physiology of an organism is in turn considered to be a pathogen. It must be noted that some species considered as destructive pathogens are not effectively killing plants, but cause damage to valuable plant parts such as cereal crops, fruit, or tree stems. According to the literature, the first description of maladies of trees was made by the Greek Philosopher Theophrastus (370–286 BCE) as contained in his book Enquiry into plants. The diseases he described were ascribed to be spontaneous and not due to external causes. However, in human medicine by the late middle ages and late 14th century, the germ theory of disease, which states that microorganisms are causative agents of disease, was proposed (https://en.wikipedia.org/wiki/Germ_theory_of_disease). The advent of novel technological advances through the invention of the microscope by Anton von Leeuwenhoek in the 17th century facilitated the investigation of microbes (bacteria) associated with higher organisms. It was not until the work of Louis Pasteur and Robert Koch on animals and humans in the late 19th century that scientifically demonstrated that disease is caused by microorganisms. However, compared to human medicine, the evidential concept of causative agents of disease and pathogenicity is relatively a recent development in the field of plant and forest pathology. Anton de Bary widely regarded as the father of modern plant pathology was the first to demonstrate that pathogenic fungi did not arise through spontaneous generation. Through elaborate experiments, he established the microbial origin of plant diseases. The German scientist and forester Theodor Hartig developed microscopic methods in the early 1800s. He observed the so-called Hartig net of mycorrhizal hyphae and that fungal hyphae are related to the degradation of wood components. He considered that hyphae are of endogenous origin in tree tissues. In the 1870s, his son Robert Hartig who is regarded in many sources as the father of forest pathology was among the first to establish wider acceptance of the idea that decay in wood is caused by microorganisms. However, Hartig (1882) himself highlights the importance of earlier work, especially the book Die Microskopischen Feinde des Waldes in 1867 by Moritz Willkomm, who made a career as the professor of botany in Tharandt, Germany, later in Tartu University, Estonia, and finally as the rector of University of Prague. The book includes detailed drawings of fungi and wood anatomy and introduces white rot and root rot of trees and advice in diagnostics (Willkomm, 1867). Hartig published the book Lehrbuch der Baumkrankheiten in 1874, followed by H. Marshall Ward’s English translation Textbook of the Diseases of the Trees in 1894 (Hartig, 1882, 1894, see also Woodward et al., 1998). Hartig was a modern scientist understanding the role of distributing scientific information. His books were printed by major publishers at the time, his laboratory hosted visiting scientists, and his major book was translated to English. It is noteworthy that Willkomm and Hartig share concerns of present-day forest pathologists such as increasing area of managed coniferous forests, new invasive diseases, and enhanced success of pathogens in managed forests. Therefore, it is natural that earlier and foundational studies set the stage for novel advances in forest pathology.

In the last 120 years, a number of milestones in the infectious diseases of trees have been recorded and forest pathologists have contributed in tackling several of such severe diseases. One of the notable diseases, chestnut blight, caused by a parasitic fungus (Cryphonectria parasitica) of chestnut trees was accidentally introduced in the early 20th century from East Asia to United States (Robin, 2001; Prospero, 2013). The disease wiped out almost all mature chestnut trees in America by 1940. In 1910, another major disease, Dutch elm disease (DED), caused by Ophiostoma ulmi spread by elm bark beetles was devastating elm trees in Europe. By 1928, it has reached North America and over a period of 40–50 years nearly killed all native populations of elm trees (Smalley, 1963). In the later part of the 20th century, the mysterious death of tanoak trees was observed in California. The causative agent of the disease was identified to be Phytophthora ramorum ascribed to be responsible for sudden oak death (SOD) of oak trees. Phytophthora ramorum has since been reported infecting a number of plants and trees in Europe (Rizzo et al., 2005; Alexander and Lee, 2010). In 1992, a number of ash trees were reported dying in Poland characterized by leaf loss and crown dieback. It was not until 2006 that the causative agent was scientifically described as Chalara fraxinea. Based on morphological and genetic analysis, the Chalara fraxinea was considered to be asexual (anamorph) stage. Consequently, the pathogen was renamed Hymenoscyphus fraxineus. The pathogen has now been reported in most of Europe (Coker et al., 2019). There are still many other forest tree diseases, which cannot be outlined here.

Despite the challenges posed by forest tree diseases, forest pathologists have contributed in tackling and mitigating the negative effects of several severe destructive diseases. A typical example is the control of chestnut blight disease using a hypovirulent isolate of Cryphonectria that host a mycovirus symbiont. A transgenic American chestnut expressing the oxalate oxidase gene has also been shown to be resistant to chestnut blight disease. Biological control has also been applied to manage other forest tree diseases such as the Dutch elm tree disease, which is controlled by injection of conidiospores of Verticillium albo-atrum strain WCS850 (marketed as DutchTrig) into the vascular system of elm trees. The spread of root and stem rot disease of conifer trees caused by Heterobasidion is successfully managed with the application of a biological control agent Phlebiopsis gigantea marketed as Rotstop during cuttings (Asiegbu et al., 2005).

3: Classification of tree disease agents: biotic and abiotic

3.1: Biotic factors

There are several biotic factors, which can affect the health of forest trees. These include pathogenic microbes (fungi, oomycetes, bacteria, phytoplasma, viruses), animals (insects, nematodes, mammals), and parasitic plants. Among pathogenic microbes (Fig. 1.1), fungi including members of Ascomycota and Basidiomycota have been primarily well-studied and reported. Insects are also one of the biotic agents; they are vital components of forest ecosystem. Many are known to have negative effects on tree health with considerable economic loss to forest industry. Several insect groups that have been reported to cause major damages to forest tree health include defoliators (e.g., Western spruce budworm, Douglas-fir tussock moth), bark borers (e.g., bark beetles, Ambrosia beetle), tip feeders, wood borers (e.g., termites), root feeders (e.g., wireworms, white grubs), and sapsuckers (e.g., aphids). Nematodes and mammals constitute another group of biotic agents that cause major damage to forest trees. Among mammals, squirrels, elk, deer, rabbits, and rodents (e.g., porcupines, gophers, beaver) are commonly reported. Parasitic nematodes have also been reported to affect a diverse number of tree species including pine, beech, teak, sandalwood, and acacia. The few parasitic nematodes that have been documented to cause diseases to forest trees are Bursaphelenchus xylophilus, Helicotylenchus, Tylenchorhynchus, Hemicriconemoides, Xiphinema, Macroposthonia, Trichodorus, Pratylenchus, Trichodorus, Meloidogyne, Subanguina chilensis, etc. (Khan, 2012). Many plants also act as parasites of forest trees; notable among them are Mistletoe (Arceuthobium minutissimum, Viscum album) https://www.britannica.com/plant/parasitic-plant) and Dodder species (e.g., Cuscuta monogyna and Carex lupuliformis) (Kaštier et al., 2018).

Fig. 1.1

Fig. 1.1 The relative size of typical pathogenic agents to a plant cell. Credit: Artin Zarsav. Modified from Agrios, G. N., 2005. Plant Pathology. Elsevier Academic Press, Boston, Amsterdam. ISBN 978-0-12-044565-3.

3.2: Abiotic factors

A number of abiotic factors can affect forest tree health. These include moisture extremes (e.g., waterlogging, drought, winter desiccation), fire (e.g., lightning, humans), high winds (e.g., windthrow), unfavorable weather, temperature, chemicals (e.g., pollutants, herbicides), and nutrient deficiencies or toxicity. Many of these factors can predispose forest trees to attack by biotic agents or cause direct damage. In practice, it may be impossible to separate primary and secondary factors, and effects of both biotic and abiotic agents are typically observed in declining forests.

4: The Disease triangle

According to Scholthof (2007), the disease triangle is a conceptual model that shows the interactions between the environment, the host, and an infectious (or abiotic) agent. Throughout their lifetime, forest trees like other plants are continually exposed to biotic agents (bacteria, fungi, oomycetes, viruses, insects, nematodes, weeds, etc.) and abiotic factors (drought, rainfall, temperature, soils, nutrients, etc). The disease triangle (Fig. 1.2A) originally conceived by George McNew in the 1960s was primarily to be used to study the interrelationship of various factors (highlighted above) in an epidemic (McNew, 1960). He defined the parameters within the disease triangle as the inherent susceptibility of the host, the inoculum potential of the parasite, and the impact of the environment on parasitism and pathogenesis (McNew, 1960). According to Agrios (2005), for a disease (Fig. 1.2B) to occur, all the elements within the disease triangle such as conducive environment, pathogen, and a susceptible host must be present. The disease triangle is therefore a classic plant pathology paradigm, and the model is equally applicable in forestry to predict epidemiological outcomes in tree health as well as develop novel ideas for forest protection. The disease triangle clearly demonstrates that all three factors are interacting with each other. However, many plant pathologists have proposed the inclusion of additional parameters such as time, vectors, and humans as contributory factors that also influence the disease outcome. The inclusion and interaction of additional causal factors were conveyed by Agrios (2005) in the disease pyramid or tetrahedron (Fig. 1.2C). The impact of human activity as the fourth factor on disease outcome may be much more prevalent in agriculture and to a lesser degree in forestry. However, a number of emerging forest tree diseases have been attributed to human impacts on forests as well as unintentional transport of plant and wood pathogens across international borders (Desprez-Loustau et al., 2016).

Fig. 1.2

Fig. 1.2 (A) The disease triangle as originally conceived ( McNew, 1960 ; Stevens, 1960). (B) An illustration of the importance of interactions between some key elements within the disease triangle for disease occurrence. (C) The illustration of disease pyramid or tetrahedron. Panel (B) modified from Scholthof, K.B., 2007. The disease triangle: pathogens, the environment and society. Nat. Rev. Microbiol. 5, 152–156 https://doi.org/10.1038/nrmicro1596.

Furthermore, in recent years, the emergence of the concept pathobiome has been used to describe that disease is not caused by a single pathogen but by a group of host-associated organisms that together reduces its health status or fitness (Bass et al., 2019).

5: Rationale for forest pathology and disease epidemiology

The diversity, adaptation and spread of pathogen population and species richness of a pathogen community may be indirectly affected by factors previously outlined above such as pathogen and host characteristics, morphological complexity and geographical distribution and range of the host. The consequent economic losses due to microbial diseases, pests, and adverse climate pose major threats to the sustainable supply of forest tree products. In addition, wood decay of standing trees in urban cities and wood in construction pose serious hazard to properties and human health. The increasing demand for wood biomass and associated products necessitates the need for concerted efforts to safeguard this valuable renewable natural resource. It is also important to systematically catalog the phenotypic and genetic diversity of tree pathogens, so they can be reliably studied.

In the process of reducing host fitness and in causing disease and mortality, forest tree pathogens play vital roles in the composition and structure of the host plant communities. Additionally, they facilitate in advancing co-evolutionary changes for adaptation and selection for resistant genotypes (Gilbert, 2002; Desprez-Loustau et al., 2016). Apart from being destructive agents, pathogens may also play key roles in the maintenance of the diversity of plant species (Bever et al., 2015). Pathogens may equally promote successional processes as well as mediate genetic diversity of host populations.

6: Nature and symptoms of tree diseases

Most tree diseases have negative effects on tree health, many of which manifest with diverse kinds of symptoms depending on the organ or parts or tissues affected. Forest pathologists have coined different terminologies to qualify several types of commonly observed disease symptoms. Most times, the name given to a particular symptom may be derived from or reflect the part of the tree affected. This is exemplified in Fig. 1.3:

Shoot blight: As shown in Fig. 1.3, it simply illustrates the death of newly emerging shoot; in some cases, the symptom is marked by the infected leaf being curled, distorted with irregular brown to black necrotic patches on the leaf.

Leaf blight: It refers to browning, yellowing, spotting, withering, and death of leaves (e.g., anthracnose disease, pine needle blight). Similar blight symptoms could also occur in other plant tissues (e.g., branches or floral organs).

Fruit spot: It is a typical symptom of diseases of fruit trees caused by microbial pathogens. Spot is usually localized and often accompanied by tissue discoloration and softening. In some cases, the spots merge together to form a rot (see fruit rot).

Fruit rot: It could start as a single spot that gradually enlarges and may often be accompanied by desiccation, wrinkling, and softening of the fruits. It is a typical disease of fruit trees (e.g., bitter rot).

Leaf spot: It is characterized by small round or uneven shaped necrotic spots or lesions on leaf surface. The discolored spots can vary in size depending on the causal pathogenic agent.

Leaf wilt: It is characterized by loss of turgor due to water loss in leaves as a result of microbial infections.

Vascular wilt: It is characterized by simultaneous death of the foliage and stem due to disruption or blockage of conducting xylem vessels or tracheids consequently inhibiting translocation of water (e.g., Dutch elm disease, oak wilt).

Crown gall: It is an uncontrolled tumor-like growth commonly observed on branches, stems, and roots of many tree species (e.g., apple, apricot, poplar, willow). Galls can be roundish or asymmetrical in shape. Crown galls are caused by the bacterium Agrobacterium tumefaciens and often range in size from a few millimeters to several centimeters and could be light-, brown-, or dark-colored.

Root rot: It is a disease where the roots of the tree succumbed to pathogen attack, resulting in decay and rot. The infected root becomes necrotic and turns black or brownish (e.g., Heterobasidion annosum root rot).

Canker: It is characterized by isolated and discolored dead tissues on the branches, bark, stems, or trunk of trees. Cankers could be depressed, swollen, flattened, circular, or irregularly shaped. Typically, canker decreases the mechanical resistance of the tree. Some cankers are lethal with potential negative impact on tree growth and productivity (e.g., apple canker, citrus canker, pine pitch canker).

Fig. 1.3

Fig. 1.3 An illustration of symptoms and kinds of tree diseases. Modified from Agrios, G.N., 2005. Plant Pathology. Elsevier Academic Press, Boston, Amsterdam. ISBN 978-0-12-044565-3.

7: The Disease cycle

The continuous sequence of events required for successful pathogen infection and disease development is called the disease cycle. This chain of interlinked events that facilitate disease development includes (a) pathogen arrival, (b) attachment/adhesion/host recognition, (c) germination/development, (d) invasion/penetration, (e) infection/pathogenesis, (f) sporulation (asexual/sexual spores), and (g) dissemination/dispersal (Fig. 1.4A) (De Wolf and Isard, 2007). For a disease to develop, the pathogen propagule must be present. The arrival or introduction of the pathogen propagule could be in the form of spores (e.g., for fungi) or bacteria cells or viruses. Following pathogen arrival, a process of adhesion of the spores to the host surface is initiated. Adhesion could be either passive or active. Passive adhesion that is reversible could be mediated through chemotaxis, spore appendages, impaction, or entrapment (Jones, 1994). Active adhesion, on the other hand, requires the secretion of adhesives and possible lectin-like interaction with corresponding host molecules (Fig. 1.4B). For fungal pathogens, successful adhesion is accompanied by spore differentiation, germination, and development of germ tube (Fig. 1.4C). Penetration of host tissues is accomplished through the formation of appressoria or penetration peg (hyphae). Apart from direct penetration through appressorium or penetration peg (hyphae), some fungi can also penetrate their host through natural openings (lenticels, hydathodes, stomata) or through wounds (Agrios, 2005). Following successful penetration, further invasive growth is facilitated by the secretion of cell wall-degrading enzymes, effectors, and production of toxins to overcome host defenses. The pathogen will reproduce and multiply on or in its host by the production of sexual and asexual spores. The spore propagules are further dispersed and disseminated by winds, animals, or vectors for the infection of another susceptible host. If all the series of stages are uninterrupted, the cycle of events leading to infection is repeated. Depending on the pathogen, the disease cycle could be monocyclic (i.e., pathogens that reproduce only one infection cycle per season) or polycyclic (i.e., pathogens that reproduce more than one infection cycle). Typically, ascomycetes and rust fungi are well-adapted to polycyclic dissemination. This facilitates exponential increase in population size over season and rapid spread of virulent genotypes. However, many other pathogens such as phytoplasma, viruses, and bacteria may use different strategies and methods for dispersal as well as to penetrate and infect their plant hosts, for example, through association with insect vectors.

Fig. 1.4

Fig. 1.4 (A) The disease cycle. (B) Active spore adhesion mediated by secreted substances (e.g., Heterobasidion pathosystem). (C) Spore differentiation accompanied by germination, development of germ tube, and invasive growth (e.g., Heterobasidion spp.).

8: Lifestyles and nutritional strategies of tree pathogens

Forest tree pathogens can be classified depending on their lifestyles and strategy for nutrient acquisition. The diverse nutritional strategies could vary from obligately biotrophic, hemibiotrophic to necrotrophic pathogenic lifestyles. Obligate biotrophic microbes are host-specialized group of pathogens that feed and depend on living tree tissues to survive and complete their life cycle (e.g., rust fungi, powdery mildew). The host response is often characterized by hypersensitive response (HR)-related cell death accompanied by the induction of reactive oxygen species (ROS) and associated with the activation of salicylic acid (SA)-dependent signaling pathway. Therefore, hypersensitive response (HR) as a form of programmed host cell death may be an effective defense against obligate biotrophic pathogens (Fig. 1.5). Hemibiotrophic pathogens unlike obligate biotrophs have an initial brief biotrophic-like phase before switching to necrotrophic growth (Mendgen and Hahn, 2002; Lee and Rose, 2010) (e.g., frosty pod rot disease on Theobroma sp. (Cacao) caused by Moniliophthora roreri). At the other extreme are necrotrophic pathogens that kill the host cells and feed on the dead tissues. Some necrotrophic fungi may also live as saprotrophs when their host is killed (e.g., Heterobasidion annosum root rot). Unlike obligate biotrophs, hypersensitive and necrotic cell death is not effective defense mechanism against necrotrophic pathogens as necrotrophs benefit from feeding on the dead host cells (Fig. 1.5). Host defenses against necrotrophic pathogens are controlled by jasmonic acid (JA)- and ethylene (ET)-dependent signaling pathways.

Fig. 1.5

Fig. 1.5 Host cell response to phytopathogen invasion.

9: Classes of resistance: Gene-for-Gene model

The outcome of the host response to pathogenic infections can be classified into two major categories: compatible interaction resulting in successful infection/disease and incompatible interaction resulting from successful host defense/resistance (Glazebrook, 2005). The susceptibility or resistance responses to pathogenic infection are indirectly a reflection of the host genetic characteristics or features. Resistance could be either host-specific resistance or non-host resistance. Non-host resistance is a broad-spectrum immune defense shown by an entire plant species to a specific parasite or pathogen (Senthil-Kumar and Mysore, 2013). This type of resistance is the most efficient and durable type of immune defense expressed by plants toward a great majority of pathogenic microbes. Host-specific resistance, on the other hand, is pathogen-specific, and it is restricted to the genotype of a specific parasite/pathogen. Host-specific resistance follows the concept of the gene-for-gene relationship (Flor, 1942). Flor (1942) demonstrated that the ability of a pathogen to cause disease and the inheritance of resistance in the host are controlled by the resistance gene (R) in the host and a parasite gene named avirulence (Avr) gene. In host-specific resistance, the variations are controlled by a single resistance gene (R). The products of resistance genes are known to interact with specific products of parasite or microbe-associated molecular pattern (PAMP, MAMP; including elicitors or effectors) produced by the pathogen (Avr) genes. The gene-for-gene model (Fig. 1.6) illustrates the ability of specific product of dominant plant resistant gene (R) to recognize corresponding products of a pathogen dominant Avr gene, by so doing confer resistance toward the pathogen. It should be noted that despite decades of research, shown examples of gene-for-gene interactions are very few, and it is likely that most of the resistance in the nature environment is a result of a number of traits.

Fig. 1.6

Fig. 1.6 The gene-for-gene model.

10: Major gene and polygenic disease resistance

Major gene or complete (qualitative) resistance conforms to the concept of the gene-for-gene relationship. In complete or qualitative resistance, one major gene determines whether the tree will be susceptible or resistant. This type of resistance is easy to screen or breed. However, pathogens with rapid evolution can easily overcome major gene resistance. This type of resistance is typically observed against biotrophic pathogens such as Melampsora spp. rusts and Cronartium spp. (Fusiform rusts). Quantitative resistance, on the other hand, refers to partial or incomplete level of resistance phenotype, which may be due to genetic variations in many causal genes (polygenic). Quantitative resistance is known to be more durable. However, the resistance is partial and the breeding can be difficult. This type of resistance is typically observed against necrotrophic pathogens such as Heterobasidion spp.

11: Hypersensitive response (HR), necrotic lesions and cell death, and reaction zones

Hypersensitive response is a feature of many biotrophic plant-microbe interactions, which leads to disease resistance. It is characterized by programmed cell death (PCD) of tissues surrounding the site of pathogen infection (Heath, 1998; Lam et al., 2001). As indicated previously (see Fig. 1.5), HR is an effective defense against biotrophic pathogens that require living tissues for their nutrition, whereas it is not an effective defense against necrotrophic pathogens for which dead cells are considered beneficial for their survival and continued invasive growth. For hemibiotrophic pathogens, HR may be an effective defense at the initial stages of pathogen infection but not in the late stages when it switches to necrotrophic nutritional mode (Balint-Kurti, 2019). Necrotic lesions or necrotic cell death, on the other hand, is a major disease symptom for many necrotrophic interactions. However, in contrast to necrotic cell death, typical morphological and physiological features of hypersensitive cell death include the activation of nucleases, fragmentation of nuclear DNA, condensation of cytoplasm, and shrinkage of cells. These biochemical features of HR also share similarities to apoptotic cell death (apoptosis) observed in animals (Heath, 1998; Lam et al., 2001).

12: Reactions zones in tree defenses

In perennial woody plants such as forest trees, defense responses to wounding and phytopathogen attack may be much more complex. Several authors have put forward different concepts and models to explain the resistance response of woody tissues. The three most widely referenced models are (1) reaction zone by Shain (1979), (2) compartmentalization of decay in trees (CODIT) by Shigo (1984), and (3) microenvironment by Boddy and Rayner (1983).

Reaction zones are necrotic regional barriers formed inwardly within tree tissues that separate healthy living xylem sapwood from the decayed brownish-colored wood. The reaction zone is characterized by increased pH toward alkalinity or neutrality due to high inhibitory levels of antifungal phenolic compounds and carbonate. The CODIT model uses a combination of features of the reaction zone together with the barrier zone for wound healing and pathogen defense. In the CODIT concept, the defense is accomplished by structural and chemical changes within what is described as four walls (walls 1–4) with special characteristics. Wall 1 is the weakest of the barriers, whereas wall 4 is considered to be the strongest of the barrier zone (for further details, see the chapter on the anatomical and molecular defense of trees in volume 3 of the Forest Microbiology book series). The microenvironment model posits that high moisture content of sapwood and low availability of oxygen are the key internal microenvironmental factors restricting invasive growth and inhibiting fungal decay of living wood tissues.

13: Wood decay on standing living trees and freshly felled trees

Wood decay is principally the breakdown of three major chemical constituents of wood (lignin, cellulose, and hemicellulose) by the enzymatic activity of microorganisms. Wood decay is ecologically important for nutrient cycling and for regeneration of young trees. Wood decay of standing living trees is often initiated through wounds, scars, openings, or exposed wood surfaces. The major agents of wood colonization and decay in standing trees and freshly felled trees are primarily fungi belonging to the groups Basidiomycota and Ascomycota.

In standing living trees, wood decay often occurs within the inner zone or heartwood of the tree (Fig. 1.7A and B). This implies that decay inside standing living trees may not be detected by visual inspection. Consequently, such trees pose a hazard to human safety as they can easily be felled by strong winds. On the other hand, wood decay or colonization of recently felled or dead trees occurs mostly in the xylem sapwood regions with heartwood not affected (Fig. 1.7C).

Fig. 1.7

Fig. 1.7 (A) Heterobasidion -induced wood decay with degradation of heartwood. (B) Invasive fungal growth on a standing living tree due to unsuccessful wound healing. (C) Colonization of sapwood of felled tree by blue stain fungi. (D) Wound healing in living trees. (E) Compartmentalization of decay in the process of wound healing.

Fig. 1.7A illustrates the nature and pattern of decay on standing trees by pathogenic basidiomycete white rot fungus Heterobasidion annosum. The decay is almost exclusively within the dead heartwood region or inner zones of the tree. This type of decay is often difficult to detect visually in the standing trees, and losses are seen at the time of cutting. There are several tools for detecting decay in standing trees in urban areas such as electromagnetic wave permittivity, ultrasonic frequency, electrical resistivity, and infrared radiation emissivity (see review by Leong et al., 2012). However, these tools may not be feasible for wood decay detection on standing trees in plantation and commercial forestry settings. An ideal rot detection approach in commercial and plantation forestry would be a tool that is cost-effective, is rapid, is accurate, and has the potential to be applied for high-throughput phenotyping or screening under field conditions.

By contrast, the sapwood of freshly cut logs or felled trees is preferably initially colonized by sugar-loving fungi that are not able to effectively decompose cellulose and lignin such as members of the fungal genus Ophiostoma as well as other fungi groups that leave dark stains on the xylem sapwood (Fig. 1.7C). The most visible type of discoloration is blue stain or sap stain. In Fig. 1.7C, it is obvious that the heartwood, perhaps due to the high concentration of toxic compounds, is not a preferred substrate for this group of sugar-loving fungi.

Although wounds are often the initial source of tree infections, in many situations, forest trees have developed mechanisms for effective wound healing (Fig. 1.7D). Naturally, trees make effort to repair wounds by either compartmentalizing or sealing the injured area. This is achieved through the formation of callus tissue to cover the injured and exposed surface (https://www.purdue.edu/fnr/extension/tree-wounds-and-healing/). The process of wound healing can take a very long time and could vary depending on the tree species, health status, and vigor. For small wounds, the tree can successfully repair and compartmentalize the wound without further visible external trace of the injury (Fig. 1.7E). In some cases, wound healing is unsuccessful resulting in infections and invasive fungal growth (see Fig. 1.7B).

14: Types of wood decay

Wood decay is primarily carried out by fungi. There are three major types of wood decay: white rot, brown rot, and soft rot. White rot decay of wood is caused by fungi belonging to the group Basidiomycota. The three major types of decay have a number of common and biochemically distinct features, which make them useful in the field identification of fungi and helpful in understanding the decomposition process. Many members of the white rot group degrade preferentially the lignin component, some degrade both cellulose and lignin, and in the process, the degraded wood becomes soft and fibrous in texture, bleached, and whitish in color (Fig. 1.8A). The white rot fungi are known to possess the capability to secrete a diverse range of lignin-modifying enzymes such as hydrogen peroxide-generating enzymes, laccases, and class II heme peroxidases (Kirk and Farrell, 1987). Typical examples of white rot fungi include Trametes versicolor (Fig. 1.8B), Heterobasidion annosum, Phanerochaete chrysosporium, Coprinellus sp., and Ceriporiopsis subvermispora. Brown rot is also caused mostly by members of the fungal group Basidiomycota and to a lesser extent Ascomycota. Brown rot fungi degrade mostly the polysaccharide components of wood (cellulose, hemicellulose) with slight modification of lignin; consequently, the decayed wood becomes brownish in color, with cubical cracks in appearance and loss of fibrous texture (Fig. 1.8C). Unlike white rotters, brown rot fungi modify lignin through non-enzymatic process by the generation of hydroxyl radicals (e.g., Fenton reaction) (Eastwood et al., 2011). Some members of the brown rot fungi have significant economic relevance due to damages to construction timber; these include Serpula lacrymans (dry rot) and Coniophora puteana (cellar fungus, wet rot). Other members of the brown rot fungal group include Postia placenta, Fomitopsis pinicola (Fig. 1.8D), and Phaeolus schweinitzii. Soft rot fungi unlike white rotters belong to the fungal group Ascomycota. Members of the soft rot fungal group preferentially attack the carbohydrate components of wood with slight modification of lignin; consequently, the decayed wood is brown or bleached in appearance. Members of this group include Chaetomium spp. and Ceratocystis spp.

Fig. 1.8

Fig. 1.8 (A) White rot decay, (B) Trametes versicolor ( white rotter), (C) Brown rot decay, (D) Fomitopsis pinicola ( brown rotter).

15: Exercises and study questions

(1)Give five examples each of white and brown rot basidiomycete fungi. You can support your examples with pictures from the nature.

(2)Identify and illustrate with pictures from your own environment typical examples of a leaf disease, canker, and fruit disease.

(3)Use a picture from your own surroundings to show abiotic disease of trees.

(4)Take a picture of wound healing in a broadleaf tree and compare the image of wound healing from a conifer tree. Is there any difference in wound healing in both different tree species?

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Chapter 2: Diagnostic tools and techniques in tree pathology

Emad Jabera; Muhammed Kashifb; Hui Sunc; Fred O. Asiegbud    a Crop Protection Department, PNG Oil Palm Research Association (PNGOPRA), Dami Research Station, Kimbe, Papua New Guinea

b Natural Resources Institute of Finland (Luke), Helsinki, Finland

c College of Forestry, Nanjing Forestry University, Nanjing, China

d Faculty of Agriculture and Forestry, University of Helsinki, Helsinki, Finland

Abstract

Diagnostic methods are vital for the study of any kind of infection of forest trees caused by biotic agents. Usually, a combination of traditional and modern verification approaches for plant pathogen identification is often deployed. This is necessary as accurate and proper identification of tree pathogens is critical for a better understanding of disease epidemiology and facilitation of better control and management measures. This chapter discusses the different diagnostic methods currently used to study pathogens causing infections in forest trees focusing primarily on microscopy, immunological, microbiological, biochemical, remote sensing, molecular methods, and sequencing.

Keywords

Diagnostic tools; Molecular methods; Microscopy; Immunology; Biochemistry; Remote sensing; Sequencing; Tree pathogens; Isolation; Identification

Acknowledgment

We wish to thank Prof. Albert Porcar-Castell and Dr. Tommaso Raffaello for useful comments and suggestions on the book chapter.

1: Introduction

Accurate and proper identification of tree pathogens is important for a better understanding of disease epidemiology and facilitation of better control measures. Early diagnosis of pathogens is vital to preserve plant biosecurity and to plan conservation strategies since pathogens play crucial role in shaping the ecological and evolutionary development in natural ecosystems (Gilbert and Hubbell, 1996, p. 98). Diagnosis of plant pathogens is initially and traditionally based on the phenotypic assessment of disease symptoms and the verification of the identity of the isolated causal microorganism in the laboratory through microscopic, cultural, and morphological approaches. Though the traditional and modern verification approaches of plant pathogen identification are reliable and robust, the methods employed are time-consuming and dependent on the experience and the skills of the experts preforming the diagnosis (McCartney et al., 2003).

Diagnostic methods are vital for the study of any kind of infection caused by biotic agents. Usually, the process starts with the visual analysis of disease symptoms at variable growth stages and from different parts of the plant including leaves, outer bark of branches, trunk, sapwood or heartwood, as well as further analysis of underground parts like roots. There are several notable fungal diseases of forest trees that have been unraveled using these approaches including foliar diseases, canker diseases, root and stem rots, vascular diseases, branch and tip blights on needle tips and cones (Gonthier and Nicolotti, 2013) as well as rust, powdery mildew, damping off, dieback, leaf spot, wilt and scab (Hussain and Usman, 2019; Jain et al., 2019; Hariharan and Prasannath, 2021). Similarly, viruses have also been identified from several plant genera including broad-leaved and coniferous forest trees. Generally, viral infections in plants can be associated with phenotypic alterations like mosaic-like leaf patterns, ring spots, lines and mottling of light and dark green, necrosis and chlorosis. Recently, European mountain ash ring spot-associated virus (EMARaV) has been classified by the International Committee on Taxonomy of Viruses (ICTV). Equally, bacterial infections have been associated with some distinct symptoms like galls, slime flux, and yellowing or browning of central core of the tree. Sometimes, bacterial diseases can have non-detectable symptoms or ultimately just marked by dying or dead trees (Gonthier and Nicolotti, 2013). Earlier, accurate and reliable diagnosis is therefore required to identify causative disease agents. Recently, certain types of sensors have been introduced to diagnose some disease-causing agents especially in case of absence of any disease symptoms. Furthermore, disease-forecasting models have also been developed to predict the disease based on occurrence and history of a plant disease in certain areas. In addition to latent infection or mild symptoms, visual disease symptoms caused by viruses, bacteria, or fungi have been well characterized using classical disease scales. Once the disease and potential causal agent have been recognized, the specimen can be observed in the laboratory for confirmation and further analysis using a combination of traditional and novel molecular methods. This chapter discusses the different diagnostic methods focusing primarily on microscopy, molecular or microbiological methods, and sequencing, which are currently used to study pathogens causing infections in plants and forest trees.

2: Traditional methods

The starting point of working with microbes is based on the principle of aseptic and sterile methods in handling of microbial cultures. The approaches involve sterilization of all kinds of glassware, tools, and media to avoid any contamination by other microorganisms, which are always found in the same environment. Generally, the basic tools required are inoculation loop, scalpel, glassware, ethanol or other disinfectants, gas burner, autoclave, and laminar flow cabinet. All kinds of glassware, water, agar media, and even contaminated objects are usually sterilized by autoclaving at 121°C for 15–30 min. Following the sterilization procedures, fungal or bacterial cultures in tubes, broth cultures, or plate cultures are transferred to new agar media plates or broth media in tubes using an inoculation loop (Pelczar Jr. and Chan, 1981; Grainger et al., 2001; Cliffe, 2016; Siddiquee, 2017).

2.1: Surface agar-based culture method

Different types of culture media are used depending on type of microorganisms and their growth requirements including nutrients, pH, temperature, and osmotic conditions. Vast majority of microorganisms are still not culturable partly due to unavailability of appropriate media composition and optimum in vitro environmental conditions (Bhattacharya et al., 2002; Basu et al., 2015), or the microbe is an obligate biotroph. Microorganisms require certain macro-elements for growth including C, O, H, N, K, S, P, Mag, Ca, and Fe, as well as microelements like Ni, Mo, Zn, Mn, Co, and Cu. These elements are used for the synthesis of various proteins, carbohydrates, lipids, nucleic acids, enzymes, and cofactors. Usually, agar media are prepared based on instructions by the manufacturer. However, the culture media can be optimized to maximize the efficient growth as required by the research experiment. Before autoclave sterilization, all ingredients are mixed well in water, and in some cases, individual media components need to be autoclaved separately. After autoclaving, it is dispensed in aliquots of 15–20 mL medium/Petri dish or in test tubes as agar slopes or slants. Agar plates or agar slants are used for fungal or bacterial growth with certain modifications and can be stored at room temperature for a few weeks (ASE, 2001; Grainger et al., 2001; Cliffe, 2016; Siddiquee, 2017).

Fungi require mainly carbohydrate and nitrogen and other macronutrients for their nutritional sources at certain pH (5–6) and temperatures (15–37°C). Generally, both complex and defined culture media are used to obtain pure fungal cultures. Usually, in vitro studies involve adding homogenized natural substrates to the media as documented in various bioassays like woody stems, leaves, seeds, corn or oat meal, and some others. The chemical composition of natural media is often not known compared to synthetic media of known formulation of carbohydrates, nitrogen, and other components. General or specific synthetic media are prepared depending on the fungal species and their growth features. Commonly used media include malt extract agar (MEA), potato dextrose agar (PDA), and several others (see Basu et al., 2015).

Similarly for bacteria pure cultures, various media compositions including simple and complex media are prepared depending on the type of bacteria and their nutritional requirements. A culture medium is considered as an enriched one when it includes all the required nutrients for bacterial growth including water, carbon and nitrogen source, and mineral salts. Moreover, a selective culture medium is composed of basic nutrients with addition of antibiotics or other chemicals to exclude contamination from other microbes (Bonnet et al., 2019). Many types of selective and differential media have been developed, which helps to differentiate diverse bacterial types. The commonly used media for bacteria are Luria-Bertani (LB) broth and agar. There are also a number of selective media for culturing bacteria phytopathogens as well as anaerobic bacteria. Plant pathogenic bacteria are usually cultivated on diverse types of media including nutrient broth-yeast extract agar, nutrient agar, King’s B medium, yeast extract-dextrose-CaCO3 agar, and medium 523 depending on bacterial strain (Mushin et al., 1959). Growth media including nutrient broth yeast (NBY) extract, Corynebacterium nebraskense or C. nebraskense selective (CNS) medium, and yeast extract dextrose selective (YDS) medium have been evaluated for isolation of bacterium from soil and plants (Gross and Vidaver, 1979). Other media like Dorset medium and Petergnani medium have also been used for cultivating mycobacteria (Wasas et al., 1998).

2.2: Pairing tests and vegetative compatibility

Vegetative compatibility is a commonly used traditional method for identification and assessing genetic diversity among populations of some fungal groups. Vegetative compatibility (VC) refers to the anastomosis of hyphal colonies from different fungal individuals to form stable heterokaryons (Leslie, 1993). Fungal individuals that fuse with one another to form heterokaryons are assigned to a single vegetative compatibility group (VCG). Vegetative compatibility is widely documented among many plant-pathogenic fungi and has proven to be a powerful tool in examining genetic diversity among their natural populations (Joaquim, 1990). VC between fungal strains is regulated by allelic or non-allelic combinations at multiple vic loci (i.e., loci that control heterokaryon compatibility) (Ford et al., 1995). The number of genetic interactions is usually increased due to the presence of multiple alleles at some vic loci. Some ascomycetes such as Podospora anserina have many vegetative compatibility groups (VCGs), and 17 vic loci have been identified, 13 allelic and four non-allelic (Glass and Kuldau, 1992). Some plant pathogenic fungi such as Verticillium dahliae have only four known VCGs (Joaquim, 1990). Fungal isolates that share the same VCG have the capability of exchanging genetic information via a parasexual process. The VCGs could be useful for monitoring isolates that are released into the environment or present in the population (Bayman and Cotty, 1991; Kedera et al., 1992). Correlations between VCGs and other traits (e.g., pathogenicity) could also lead to useful diagnostics tool for disease detection (Baayen and Kleijn, 1989; Correll, 1986; Manicom, 1990). It is often hypothesized that some pathogens are found in one or only a few VCGs. Consequently, a pathogen can be diagnosed through placement into a particular VCG rather than through laborious pathogenicity tests (Puhalla, 1985). The traditional methods of inoculating plants and fulfilling Koch’s postulates are time-consuming; therefore, pairing an unknown plant pathogen with a tester strain of a known VCG could be much simpler and quicker way to ascertain the identity of the infecting fungus (Leslie, 1993). There are several methods to identify VCGs based on heterokaryon formation, such as barrage formation test, direct heterokaryon formation observation, and partial diploids test (Leslie, 1993; Puhalla and Hummel, 1983; Strom and Bushley, 2016). The barrage or demarcation zone formation (Fig. 2.1) relied on the vegetatively incompatible strains and may take the form of opaque, a pigmented or clear zone between two strains (Aanen et al., 2010). The absence of a barrage indicates the vegetative compatibility. The direct test of heterokaryon formation involves the establishment of a stable heterokaryon, in which auxotroph or pigmentation markers were normally applied due to distinguishing colony. The introduction of nitrate non-utilizing (nit) mutant test makes this observation easier (Puhalla, 1985). The mutants are isolated based on their inability to reduce chlorate to chlorite, a compound toxic to fungi and this has become a standard test for grouping fungal isolates into vegetative compatibility groups (VCGs) (Puhalla, 1985). The toxicity of chlorate is due to nitrate reductase activity. Nitrate reductase reduces chlorate to more toxic chlorite, whereas nitrate reductase negative mutants can tolerate higher concentrations of chlorate, and this principle is used for the selection of nit mutants.

Fig. 2.1

Fig. 2.1 (A) Pairing test of two Heterobasidion parviporum isolates that are vegetatively incompatible showing a clear barrage/demarcation zone. (B) Microscopic view of clamp connection (the loop shaped structure), typically observed in Basidiomycetes fungi.

However, there have been some doubts on the utility of VCGs in disease diagnosis. There are possible errors in diagnosis. For example, the VCG test could detect artificially high levels of the pathogen if a non-pathogenic strain(s) is in the same VCG as the pathogenic strain. Secondly, strains belonging to the same pathogenic subgroup would belong to many VCGs, indicating that some non-pathogenic strains may be accompanying one or a few pathogenic strains in their attack on the host ( Ghag et al., 2015; Talma et al., 1991). Another drawback with VCG analysis is the ability to form weak heterokaryons by members of different VCGs (Talma et al., 1991). Papaioannou and Typas (2015) noted that some of these weak heterokaryons could be short-lived and unstable. The results however demonstrated the potential evidence of exchange of genetic material between members of different VCGs. Similarly, forest pathogen Heterobasidion species complex has shown variable somatic compatibility including H. parviporum, H. abietinum, and H. occidentale species across North America, Asia, and Europe with exceptionally high compatibility between geographically closely related populations and found lowest between North American and European isolates (Korhonen and Hintikka, 1980; Korhonen and Stenlid, 1998; Stenlid and Karlsson, 1991; Dai et al., 2003). In Heterobasidion species, heterokaryon isolates are usually determined by one of the prominent cytological features found in Basidiomycetes known as clamp connections (Fig. 2.1B). These clamps are formed by combination of two different nuclear haplotypes which transform into a secondary heterokaryotic mycelium (Johannesson and Stenlid, 2004; Vainio et al., 2015).

2.3: Single spore isolation

Obtaining a pure culture is vital for identification of fungal species which is mainly achieved by single spore isolation using simple techniques. The procedure described here was developed for Heterobasidion and might need to be adjusted for other species of fungi. Briefly, the freshly cultured fungal isolate (about 2–3 weeks old) in Petri dishes (diameter 9 cm) on solid media (e.g., malt extract agar MEA) is used. The spores are washed from the agar surface with several aliquots of sterile water with gentle agitation of the colony. The resulting spore suspensions are diluted in 1-l sterile water followed by shaken, and aliquots of 0.5 mL can be spread evenly on MEA plates. Germinating spores could be checked under a microscope after 36-h incubation at room temperature. Single spore can be picked with glass capillary under microscope to ensure that only one spore is selected (Sun et al., 2009).

2.4: Spore count by hemocytometer

Hemocytometer is a classical method used to determine the number or concentration of fungal spores (Valdez and Piccolo, 2007; Singh and Singh, 2007). This has been a traditional procedure for direct counting of spores (Heagle and Moore, 1970; Clifford, 1972; Asher et al., 1982). For this purpose, spore suspension on a hemocytometer is prepared. The coverslip on the hemocytometer is placed, and certain amount of the cell suspension depending on type of hemocytometer and fungi is pipetted in one of the counting chambers. The well-mixed suspension is further spread to chambers carefully without overfilling. The spores are counted in each of the four 0.1-mm³ corner squares under a microscope. Recently, different spores including conidia, ascospores, and urediniospores related to cultures of diverse fungal species such as Heterobasidion annosum, Magnaporthe oryzae, Leptosphaeria maculans, and Kabatiella caulivora have been estimated by suspending in 0.001% Tween-20. The desired concentration for plant infection is often leveled to 10⁶ spores per mL and is estimated by using a hemocytometer (Barua et al., 2019). To achieve a reliable spore suspension on a hemocytometer, each concentration should be based on an average of three replicates. Although this technique is fairly widely used, it should be noted that many well-equipped labs possess automatic cell counters that allow to determine spore concentrations much faster.

2.5: Microscopy examination

Traditionally, microscopy analysis is conducted to make a preliminary identification of fungal pathogen. The fungal hyphal growth is observed under microscope. Microscopical observation of morphological and reproductive structures of fungi present on symptomatic plant or tree materials is often performed. The nature of asexual structures (conidia, pycnidia), sexual fruiting bodies, and the dimensions of both sexual and asexual spores could yield useful information for preliminary identification. A microscope eyepiece fitted with an ocular micrometer (https://en.m.wikipedia.org/wiki/Ocular_micrometer) could be used to measure spore dimensions. Phase contrast microscopy (https://en.wikipedia.org/wiki/Phase-contrast_microscopy) as well as fluorescent and confocal laser scanning microscopy (see Kuo et al., 2014) can be used to visualize fungi on plant surface. In addition, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) could be deployed for additional histological analysis of infected tissues (see Asiegbu et al., 1993 and Asiegbu et al., 1994 for detailed sample preparation methods for SEM and TEM).

2.6: Koch postulates

The isolated and identified pathogen isolate must fulfill Koch postulates in order to be considered a causative agent of the tree disease. The Koch postulates are based on the work of Robert Koch. In 1882, Robert Koch published his discovery of the tubercle bacillus (Mycobacterium tuberculosis) which led him to frame the postulates, and received the Nobel Prize in Medicine in 1905. The Koch postulates have been widely used to elucidate many diseases of plants and trees. The four postulates include the following:

i.The same organism must be present in every case of the disease.

ii.The organism must be isolated from the diseased host and grown in pure culture.

iii.The isolate must cause the disease, when inoculated into a healthy, susceptible host.

iv.The organism must be reisolated from the inoculated, diseased host.

It is however important to note that the Koch postulates may not apply to unculturable organisms.

2.7: Biochemical methods (bacteria and fungi)

Phospholipid fatty acid (PLFA) profiling of microbial agents has been developed for a quantitative analysis of fungi and bacteria in environmental sample. Fatty acids are specifically extracted from environmental samples by using organic solvents followed by fractionation of lipids into neutral, glycol-, and phospholipids. Later, they are further fractionated into alkaline methanolysis to synthesize fatty acid methyl esters (FAMEs) which are further analyzed based on gas chromatography–mass spectrometry (White and Ringelberg, 1998). A PLFA sample profile describes the quantification of each of the PLFA fraction which also depends on the variation of fatty acids found in cell membranes of microbes. Formulation of certain PLFA profiles is described by variable chain length, saturation, and branching of fatty acids. Accordingly, these profiles can be employed as fingerprints of particular microorganisms (Leckie, 2005; Steer and Harris, 2000; Willers et al., 2015). Moreover, there are several factors including metabolic state of the microbial agent, environmental conditions, and their exposure to toxic substances that may influence PLFA profiles (Kieft et al., 1994; Mahmoudi et al., 2013; Reinsch et al., 2014). Consequently, PLFA analysis has several applications for determination of microbial biomass in the environmental sample (Willers et al., 2015).

2.8: Specimen and pathogen isolate preservation

Different methods are adapted to store and maintain the specimen and viable cultures of fungi for either short or long period of time (Box 2.1). Available methods include storage in sterile water, storage under paraffin or mineral oil, cryopreservation in liquid nitrogen with and without solid particulate carriers, freezing at −  20°C or −  80°C, and freeze-drying (Borman et al., 2006). Isolates preserved in liquid nitrogen and in lyophilized form are widely accepted to represent those methods of storage, which best minimize genetic alterations. Many of these methods may not be widely available in all fungal biology laboratories. Isolates are in addition stored as suspensions of fungal spores and mycelium for relatively short periods in sterile distilled water at room temperature (Borman et al., 2006).

Box 2.1

Prior to storage, the purity of cultures must be checked along with other morphological features. The following preservation methods are commonly used ():Dhingra and Sinclair, 1995

(1)The samples can be stored from few weeks to months in cold temperatures +  4°C depending on type of specimen or isolated pathogens.

(2)Freezing liquid nitrogen is also one of the common methods to preserve plant pathogen samples. The organisms can be stored in −  20°C to −  80°C. The viability of cells is more sensitive in 0°C to −  20°C, mostly due to freezing and thawing shocks and storage time. However, storage efficacy of deep freezing depends on type of organism and medium. Generally, bacteria are grown in broth culture at optimum conditions. After centrifugation, the cells in pellet are washed with phosphate buffer (pH 7). Cells are resuspended in phosphate buffer with 15% glycerol and sealed followed by storage in −  20°C to −  80°C. Similarly, fungal spores are stored and can be viable for up to 6 months storage and sometimes a few years at −  80, if vials are not subjected to freezing/thawing cycles. Moreover, liquid N2 (−  196°C) storage which causes the metabolic activity of most microorganisms to almost stopped. Fungal samples (spores or mycelia) are stored in liquid N in glycerol, glucose, sucrose, or DMSO; however, samples become sensitive to other storage methods like lyophilization.

(3)Mineral oil is also used to cover, when fungi and bacteria are grown on agar media for longer viability for weeks to several years.

(4)Drying plant pathogens in their host tissues or agar cultures can preserve samples for long time. The conditions for dried storage method can be