Bacterial Diseases of Crops

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Bacterial Diseases of Crops: Elucidation of the Factors that Lead to Differences Between Field and Experimental Infections Jay Ram Lamichhane INRA, UAR 1240 Eco-Innov Research Unit, Thiverval-Grignon, France E-mails: [email protected]; [email protected]

Contents 1. 2. 3. 4.

Introduction Presence of Inter- and Intraspecies Bacterial Cooperation Frequent Climatic, Edaphic, and Nutrient Stresses Existence of Biotic and Abiotic Associations that Lead to Complex Disease Phenomena 5. Occurrence of Simultaneous Biotic Stresses 5.1 Different Time of Symptom Appearance 5.2 Masking Phenomena 6. Higher Availability of Ports of Entry and Putative Infected Sites 7. Difference in the Growth Stage of Plant Materials Infected 8. Conclusion Acknowledgments References

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Abstract Diseases caused by plant pathogenic bacteria have attained great concern worldwide as they are responsible for severe economic losses throughout the cultivated areas. Although studies performed in experimental conditions have provided many new insights into chemical and molecular signaling between plants and bacterial pathogens during pathogenesis, little is known about the factors that interact in natural field conditions. In particular, a wide gap exists between these two systems in terms of disease occurrence and severity. This review attempts to highlight the possible reasons that make natural field conditions different from the experimental ones, which might be useful to bridge the current gap and to facilitate the development of adequate control measures. Advances in Agronomy, Volume 134 ISSN 0065-2113 http://dx.doi.org/10.1016/bs.agron.2015.06.006

© 2015 Elsevier Inc. All rights reserved.

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1. INTRODUCTION The occurrence and evolution of bacterial diseases are very complex processes in natural field conditions, which are influenced by dozens of abiotic and biotic factors. The difficulty of dealing with these factors has limited our understanding about their hidden role, the interactions that occur among them, and the consequences in crop health. Most experimental studies performed to date focused solely on the interactions in sensu stricto between the host and individual stress factor (climatic, biotic, and edaphic) by increasingly widening the difference between what happens under field conditions and what is generally observed in experimental studies (laboratory, growth chamber, and greenhouse). In this review, an attempt is made to discuss about some putative factors that could play a crucial role in the occurrence and development of plant diseases under field conditions with particular emphasis on how the influence of such factors make unique field epidemics. Most plant pathologists wonder about marked differences between field and laboratory infections, which is difficult to answer even after several decades of experience on a given pathosystem. In particular, the different extent of damage caused by a given bacterial pathogen between the two systems raises several questions. Hence, a considerable gap might exist between the knowledge gained by experimental studies and the knowledge required to develop plants with resistance or enhanced tolerance to field conditions. This gap might explain why a given plant genotype that is resulted resistant and/or moderately resistant in the laboratory to a particular biotic stress (i.e., infection due to pathogens) fails to show resistance when tested under field conditions. It means that currently used techniques for developing and testing stress-resistant/ tolerant plants through individual stress factor, applied one at a time, is likely inadequate (Mittler and Blumwald, 2010). A focus on different aspects of field conditions is thus needed to bridge this gap and to facilitate the development of adequate control measures. Overall, six major factors can differentiate natural field infections from what occurs in experimental studies: (a) presence of inter- and/or intraspecies bacterial cooperation, (b) frequent climatic, edaphic, and nutrient stresses, (c) existence of biotic and abiotic associations that lead to complex disease phenomena, (d) occurrence of simultaneous biotic stresses, (e) higher availability of ports of entry and putative infected sites, and (f ) difference in the growth stage of plant materials infected. An attempt is made here to

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explain the role played by each of these factors including evidences found throughout the literature works.

2. PRESENCE OF INTER- AND INTRASPECIES BACTERIAL COOPERATION The central dogma of plant pathology is based on the plant disease triangle. This belief considers that the development of a given disease requires three major factors, a host plant, an aggressive pathogen, and a conducive environment. In cases in which a given disease is disseminated by an insect vector, the concept of disease pyramid has been extended to a fourth factor (Purcell, 2013). To date, the classical vision is based on the fact that bacterial diseases, as most other microbial diseases, result from the ability of a given bacterial pathogen to colonize and regulate gene expression in response to the host environment. For this reason, many studies yet focus on the molecular interactions of monoculture infections whereby only one pathogen is considered. However, microbes generally inhabit complex polymicrobial communities where interactions between individuals shape the common and biological activities of the population (West et al., 2007). Indeed, a recent study reviewed a large number of plant diseases due to polymicrobial infections that are increasingly reported in the literature (Lamichhane and Venturi, 2015). Such polymicrobial diseases occur through a complex interaction between pathogenecommensal (Buonaurio et al., 2015) and pathogenepathogen (Lamichhane and Venturi, 2015) thereby leading not only to disease occurrence but also to increased disease severity. For this reason, a fifth and perhaps one of the most important factors should be included to the disease triangle, which can be called as “cooperation among microorganisms.” Crop disease epidemics caused by bacteria are thus likely to occur due to the result of complex multispecies bacterial cooperation. However, under field conditions, still there might be a large number of pathogenic and nonpathogenic bacterial species in association with plants, poorly known to date. Not all bacterial species can be easily isolated or cultured, which limit our understanding of the overall bacterial community. Taken together, epiphytic, endophytic, and rhizosphere bacteria in a single host plant are not restricted to a single species but comprise large multispecies communities that interact on/in plants. Consequently, beneficial and/or harmful effects on plants are the combined outcome of their complex interactions. Once

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the infection occurs on a host plant, most of these species might contribute directly or indirectly to symptom development leading to a severe form of infection, often evident under field conditions. Plant pathologists are aware of the fact that the severity of diseases reproduced by the pathogenicity tests under experimental conditions has little to do with what occurs under field conditions. In some cases, it is difficult to reproduce disease symptoms under experimental conditions. This is especially true when working with strains of bacterial species belonging to Pseudomonas, Xanthomonas, and Erwinia. By contrast, the soilborne pathogen Ralstonia solanacearum represents an exception in this regard, although reproduction of symptoms in experimental conditions also depends on the aggressiveness of a given strain. In studying bacterial canker of hazelnut, Vuono (2005) was not able to induce disease symptoms under experimental conditions with Pseudomonas syringae strains isolated from diseased hazelnut plants. Inoculation of plants with different techniques and incubation of plants under different conditions did not allow the author to fulfill Koch’s postulates. Attempts were made several times over the years without any success to reproduce symptoms, although the original strains were constantly reisolated from the inoculated point. A recent study further confirmed that the symptoms reproduced on hazelnut plants by artificial inoculation of P. syringae were very weak (Lamichhane and Varvaro, unpublished data). In addition to P. syringae, the authors isolated several other bacterial species from the symptomatic plant tissues affected by sudden hazelnut decline suggesting that the disease could be caused by the interaction of a wide range of bacterial species. Similarly, in studying apple fire blight, caused by the pathogen Erwinia amylovora, several authors failed to produce infections under experimental conditions especially when they inoculated plants without causing any lesion (Brooks, 1926; Crosse et al., 1972; O’Gara, 1912; Pierstorff, 1931). The reason behind the failure to confirm Koch’s postulates under experimental conditions could be due to the absence of other cooperative bacterial species, whereas under field conditions, often diseases might have occurred in cooperation with numerous microbial species. On the other hand, one can wonder whether only plants under field conditions bear epiphytic, endophytic, and rhizosphere microflora. The lack of comparative studies in the literature does not allow answering this question. However, it can be assumed that plants grown in controlled environments cannot harbor microorganisms that arrive through air mass, water,

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or insect vectors. Likewise, the rhizospheric soil might harbor much lower populations of microbes under controlled conditions, in which small potted plants are used in sterile and often uniform substrate, compared to the field conditions whereby very high microbial diversity exists in the rhizosphere (Peiffer et al., 2013; Teixeira et al., 2010). In addition, the role of such microbes at the level of phyllosphere, rhizosphere, and endophytic surface is the result of a long-term interaction under field conditions (months, years, decades, or even centuries for woody plants) compared to those of experimental ones whereby plants are maintained only for a short period of time (days or months). Such phyllosphere, endophytic, and rhizosphere bacterial communities change continuously during plant growth over time in the field (Bulgari et al., 2014; Redford and Fierer, 2012; Sugiyama et al., 2014).

3. FREQUENT CLIMATIC, EDAPHIC, AND NUTRIENT STRESSES Besides biotic stress, plants under natural conditions are exposed to a large number of climatic and/or edaphic stresses. Major stresses include heat/drought, late frost, extreme soil pH values and nutrient imbalances. Some of these stress factors may fluctuate significantly in intensity and duration on time scales of hours, days, seasons, or years; others may change slowly and gradually affect plant growth conditions. The occurrence of one or more stresses, alone or simultaneously in the field, can predispose plants to biotic stress, which might result lethal to crops (Atkinson and Urwin, 2012). Increasing evidences suggest that climatic stresses are conducive to the occurrence and severity of diseases. Summer heat and/or water stress predispose woody plants to canker diseases caused by facultative parasites (Bier, 1959; Worrall et al., 2010). Late-season water deficit predispose a large number of plant species to pathogen attacks (Bertrand et al., 1976; Guyon et al., 1996; Marie-laure and Benoit, 2006). Schoeneweiss (1981, 1975) described how environmental stresses, in particular water stress (drought) and freeze, predispose woody plants to the attacks of secondary pathogens. Field disease incidence of hazelnut bacterial blight disease increased with increasing rainfall and soil nitrogen content (Lamichhane et al., 2013). Also, frost events followed by freezing and thawing phenomena, caused by severe fluctuations of day/night temperature, are reported to trigger bacterial infections on a variety of woody crop species, worldwide, leading sometimes to severe plant disease epidemics (Ferrante et al., 2012; Lamichhane et al., 2013; Vigoroux, 1989; Zhao et al., 2013).

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In today’s intensive monoculture condition, a range of different problems exists in soil such as overexploitation, salinity, acidification, and contamination by various pollutants that are harmful to crop health (Matson et al., 1997; Singh, 2000). Soil contamination by herbicides, pesticides, heavy metals, and other toxic aromatic compounds provoke direct damage to plants by causing stress once they are assimilated and on the other hand they can influence the beneficial microflora associated with plants thereby altering their balance (Glassman and Casper, 2012; Siddiqui and Ahmed, 2006). Plant pathogens such as P. syringae seem to have acquired the capacity to metabolize aromatic compounds (Bartoli et al., 2015), which might be the result of long-time heavy use of herbicides (e.g., glyphosate or atrazine). The capacity of these pathogens to metabolize aromatic compounds represents a serious threat to the durability of agriculture since such compounds (e.g., phenolic substances) are generally produced by plants for their defense followed by pathogen attacks. In this way, pathogenic bacteria may significantly increase their fitness at the expense of beneficial one thereby altering crop health conditions. A similar scenario can be proposed for the copper-resistant strains of bacterial pathogens, which has become a serious problem in agriculture due to the development of resistance to this heavy metal. Overall, such risks are strikingly higher to woody plants than their herbaceous counterparts because woody plants have a long life span during which they are exposed to any alteration that occurs in their habitat. Mineral nutrients are essential for the growth and development of plants and microorganisms, and are important factors in plant-microbe interactions (Datnoff et al., 2007). Each nutrient affects a plant’s response to disease, either positively or negatively, which is unique to each plant-disease complex. Plant nutrients may affect disease susceptibility through plant metabolic changes, thereby creating a more favorable environment for disease development. Once a pathogen infects the plant, its physiology becomes alerted especially with regard to mineral nutrient uptake, assimilation, translocation, and utilization (Huber and Graham, 1999). Pathogens may immobilize nutrients in the infected tissues or interfere with translocation or utilization of nutrients, inducing nutrient deficiencies or toxicities in plant. Soilborne bacterial pathogens commonly infect plant roots, reducing the plant’s ability to take up water and nutrients. The resulting deficiencies may lead to secondary infections by other pathogens. Vascular bacterial pathogens infect the plant’s vascular system and impair nutrient or water translocation. Such infections can cause root starvation,

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wilting, and plant decline or death, although the pathogen itself may not be toxic and/or harmful. A balanced nutrient availability is indispensable for plant growth and is considered optimal for disease resistance. Overall, plants with an optimal nutritional status have the highest resistance (or tolerance) to diseases (Huber and Graham, 1999). Once the nutrient availability deviates from the optimal level plant susceptibility to diseases begins to increase (Graham, 1983). On the other hand, excessive presence of a given nutrient not only leads to toxic effects but also inhibits the assimilation of the others. Establishment of a balanced nutrient supply is not always an easy task under field conditions since nutrient content in soil varies with soil type, texture, and also the type of soil management. The optimal quantity to be supplied is difficult to calculate in open field conditions whereby nutrient leaching is frequent. Throughout the field, different nutrient-related conditions cause plant stress thereby favoring pathogen attacks. Summer heat increases the transpiration rate, which could result in enhanced uptake of salt or heavy metals leading plants to stress. A higher availability of nitrogen and a lower presence of magnesium favor bacterial disease development (Snoeijers et al., 2000). By contrast, limited availability of key mineral elements in soil such as iron, copper, zinc, or manganese could result in an enhanced oxidative stress in plants (Ranieri et al., 2001). Such micronutrients are required for the function of different defense enzymes, such as superoxide dismutase or ascorbate peroxidase (Tsang et al., 1991). Similarly, exposure of plants to excess concentrations of redox active heavy metals such as iron and copper results in oxidative injury (De Vos et al., 1992; Gallego et al., 1999; Mazhoudi et al., 1997). In some cases, the presence at very high concentration of a mineral ion results in a complete suppression in uptake mechanism of the other (Pathak and Kalra, 1971). For example, the susceptibility of peach to bacterial diseases was influenced by the application of calcium, nitrogen, and indoleacetic acid (Cao et al., 2006). Soil pH is another factor that markedly affects plant health. Soils with lower pH values can increase the susceptibility of fruit tree species to P. syringae diseases of stone fruits (Melakeberhan et al., 1995). Similarly, soil texture and organic matter content affect the severity of bacterial diseases. Organic amendments improve soil functions such as infiltration, water-holding capacity, nutrient retention, and release allowing root system to expand. The presence of compact clay soil hinders root system expansion causing water stress and predisposing plants to pathogen attacks (Lamichhane et al., 2013; Matthee and Daines, 1968).

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4. EXISTENCE OF BIOTIC AND ABIOTIC ASSOCIATIONS THAT LEAD TO COMPLEX DISEASE PHENOMENA One of the most important findings in plant pathology has been the demonstration of many complex associations between plant pathogens, insects, and nematodes. Various forms of associations may assume among these organisms, ranging from simple antagonism and parasitism to more intimate associations of pathogenesis and endosymbiosis. Such associations among biotic, climatic, and edaphic factors significantly influence host colonization. This appears to be the case particularly with stressed plants, which are susceptible to multiple infections by opportunistic pathogens such as P. syringae and Xanthomonas arboricola (Lamichhane, 2014). Many complex associations between nematodeepathogen (bacteria, fungi, and virus) disease complexes reported on a variety of crop species suggest the frequent occurrence of complex disease phenomena in the field (Back et al., 2000; Bertrand et al., 2000; Brown et al., 1995; Cao et al., 2006; Schellenberger et al., 2011). Experiments conducted under carefully controlled conditions also confirmed such associations. Nematodeebacteria interaction is particularly important because nematodes may carry a diverse bacterial population that could potentially influence pathogen evolution. The evidence that soft rot enterobacteria such as Pectobacterium carotovorum colonize nematodes and use them as vectors suggests the importance of nematodeeplant pathogen interaction in plant disease epidemiology (Nykyri et al., 2013). Under field conditions, the bacterial-feeding nematodes may transport bacteria to a relatively short distance, such as between neighboring potato tubers or plants or from unrotten plant remnants to healthy plants in arable land. Synergistic relationships between plant pathogenic bacteria and nematodes in increasing the severity of plant diseases (Cao et al., 2006) likely began a long time ago. Comprehensive genomic analysis (Bird et al., 2003; Scholl et al., 2003) revealed a surprising number of nematode genes as being candidates for having arisen via horizontal gene transfer from bacteria. This finding suggests that associations between nematodes and bacteria may have been extensive in the evolutionary past. Numerous studies pointed out that under field conditions nematodes often lead to increased disease severity caused by both soilborne and nonsoilborne plant pathogenic bacteria. Examples are P. syringae caused bacterial canker on peach, plum, prune, and almond (Cao et al., 2006) and R. solanacearum caused bacterial wilt on solanaceous crops (Deberdt et al., 1999). Although little is known of the underlying

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mechanism of interactions between nematodes and plant pathogenic bacteria, it has been reported that nematodes as primary pathogens create ports of entry inducing wounds, induce cellular modifications in plant tissues, decrease leaf concentrations of nutrients, increase water stress, and induce imbalance of growth hormones (Cao et al., 2006; Napiere and Quimio, 1980). Of equal importance is the association between plant pathogenic bacteria and insects. Generally, the dependence of many insects and phytopathogens on plants as their primary source of nutrition may lead to an overlap of ecological niche, providing the necessary conditions for insects to encounter, contact, or ingest phytopathogenic bacteria. Repeated encounters of phytophagous insects with phytopathogenic bacteria that reside in or on their preferred host plants are very common under field conditions. Phytopathogenic bacteria have evolved to harness insects as vectors to affect their dissemination and delivery directly on or into their preferred plant hosts (Nadarasah and Stavrinides, 2011). The role of insects as vectors of phytopathogenic bacteria has been demonstrated in kiwifruit, P. syringae; grapevine, Xylella fastidiosa; corn, Pantoea stewartii; cucurbits, Serratia marcescens; cucurbits, Erwinia tracheiphila; pome, E. amylovora; citrus, Candidatus liberibacter, and potato, P. carotovorum pathosystems (Nadarasah and Stavrinides, 2011; Pattemore et al., 2014).

5. OCCURRENCE OF SIMULTANEOUS BIOTIC STRESSES Under field conditions, simultaneous attacks of different pathogens can be observed on the same plant thereby leading to severe damage. Regardless of any cooperation between these pathogenic agents, such damage results due to the cumulative effect of biotic stresses. An example is tomato in which 74 pathogenic species (4 nematodes, 8 bacterial, 17 viral, and 45 fungal species) can cause disease (http://en.wikipedia.org/ wiki/List_of_tomato_diseases). The life span of tomato under field conditions is approximately 4 months. Tomato represents one of the many examples that explain the impressive disease pressure on plants grown today under intensive monoculture. Although all of the aforementioned diseases on tomato are less likely to occur in a single growing season, most of these pathogens can be simultaneously detected on this crop in particular climatic conditions. Hence, the putative risk of pathogen attacks in a given crop under field conditions is particularly alarming. In such conditions, plant

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defense system may fail to promptly react to simultaneous attacks of different pathogens leading to severe infection and consequent yield losses. The recognition of disease symptoms through simultaneous infections caused by plant pathogens on the same plant is often difficult for the reasons described below.

5.1 Different Time of Symptom Appearance Based on what pathogen we are looking for, conditions for disease development can markedly differ throughout the year. Both of the bacterial pathogens P. syringae (Lamichhane et al., 2014) and X. arboricola (Lamichhane, 2014) can infect and cause disease on Prunus spp. (stone fruits and almond). Both bacterial species have an epiphytic phase on Prunus spp. and both are capable of causing disease on leaves and woody tissues. The first is psychrophilic bacterium while the second is mesophilic. Consequently, infections of P. syringae begin from early spring to early summer while those of X. arboricola are commonly observed from early to late summer. Rarely, there is an overlapping infection and subsequent symptom developments on Prunus spp. Likewise, on hazelnut, economically important bacterial diseases are sudden decline and bacterial blight (Lamichhane et al., 2013; Lamichhane and Varvaro, 2014) caused by P. syringae and X. arboricola, respectively. On hazelnut, P. syringae does not have an epiphytic phase nor does it cause leaf symptoms while X. arboricola has an epiphytic phase and causes leaf spots. Both pathogens infect woody tissues on hazelnut (Lamichhane and Varvaro, 2014), although the time of symptom appearance may differ.

5.2 Masking Phenomena The aggressiveness of a given pathogen and the extent of damage it causes on a given plant are variable under field conditions. Consequently, mild presence of infections caused by less aggressive pathogens can be easily masked on plants by the severe infections caused by aggressive pathogens. Some plant species are attacked by a surprising number of bacterial pathogens like those described for tomato in which P. syringae causes bacterial speck and spot (Lamichhane et al., 2015), Xanthomonas campestris pv. vesicatoria causes bacterial spot (Potnis et al., 2015), Clavibacter michiganensis subsp. Michiganensis causes bacterial canker (De Leon et al., 2011; Lamichhane et al., 2011), and R. solanacearum causes bacterial wilt (Genin, 2010). The first two bacterial species are epiphytic pathogens and cause similar symptoms on leaves (P. syringae causes occasionally also necrotic spots on fruit) in

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mild form and are often interchangeable unless an accurate diagnosis is made. By contrast, C. michiganensis and R. solanacearum are aggressive vascular pathogens. When tomato plants harbor all of these pathogens and simultaneously get infected, mild infections caused by epiphytic pathogens can be completely masked by canker and wilting caused by severe vascular pathogens. Although simultaneous infections by these pathogens are less likely to occur for the reason mentioned above (different optimal temperature for growth), symptoms recognition would be difficult under such circumstances. In some cases, the same disease with identical symptoms can be caused by different bacterial pathogens alone or in association. On rice, two bacterial species (Burkholderia glumae and Pseudomonas fuscovaginae) are present alone or in association with sheath rot and grain discoloration (Cottyn et al., 1996). Similarly, pith necrosis of tomato can be caused by nine bacterial species alone or in association (K udela et al., 2010; Moura et al., 2005; Saygili et al., 2008). The use of selective or semiselective media for the detection of such pathogens can mask the presence of one or the other depending on the type of medium used. On the other hand, the use of nonselective medium could equally be a problem for an effective diagnosis given that some of these species (P. fluorescens, Pectobacterium atrosepticum, and P. carotovorum) grow very rapidly thereby masking the presence of other slow-growing species. Although several advanced culture-independent techniques, such as next-generation sequencing, allow identification of bacterial species without being isolated on the media (Nikolaki and Tsiamis, 2013), the isolations of such microbes result essential to perform the pathogenicity tests. The association of different bacterial phylogroups belonging to the same species in causing disease is frequent under field conditions. Two different phylogroups of P. syringae (namely, pv. papulans and pv. syringae) can be frequently found in association with both symptomatic and asymptomatic apple bud tissues (Burr and Katz, 1984). Likewise, six different P. syringae phylogroups (namely, P. amygdali, P. viridiflava, and P. syringae pvs. avii, cerasicola, morsprunorum, persicae, syringae) cause disease on Prunus spp. and often they are associated with epiphytic and endophytic parts of both healthy and infected plants (Lamichhane et al., 2014). On kiwifruit, three different phylogroups are associated with symptomatic or asymptomatic plant parts (Lamichhane et al., 2014). The presence of mild disease symptoms on these hosts, caused by less aggressive strains, can be masked by severe diseases caused by the vascular pathogens, which are common under field

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conditions. Furthermore, the presence of pathogenic species can mask that of nonpathogenic ones given that the latter are often slow growing and are difficult to culture. A wide population of pathogenic and nonpathogenic P. syringae strains found in association with different plant species confirms this hypothesis (Malvick and Moore, 1988).

6. HIGHER AVAILABILITY OF PORTS OF ENTRY AND PUTATIVE INFECTED SITES Bacterial pathogens are not able to access into plant tissues directly and for this reason they require either natural openings, such as water and gas pores (stomata, lenticels, hydathodes, and trichomes) or wounds created mechanically (pruning, hailstones, lesions created during treatments, etc.) (Lamichhane et al., 2014; Lamichhane et al., 2015; Lamichhane, 2014). Under field conditions, there are often favorable situations where pathogens can get access through natural openings such as hydathodes, which often remain open, or stomata which open during the day under certain conditions. For example, during heat stress, plants open their stomata to cool their leaves by transpiration, which represent an ideal port of entry for bacterial pathogens to enter. Once the pathogen gets access, it starts to proliferate in the intercellular space prior to beginning the infection process. In addition to the natural openings, the risk of lesion formation and the number of potential lesions per plant are much higher under field conditions. Examples are insect chewing, hailstone, pruning, and trimming. Such lesions represent an ideal port of entry for bacterial pathogens. Under optimal conditions of temperature and humidity, pathogenicity tests on annual plants with strains of foliar bacterial pathogens show a gradual development of symptoms until 10 days post inoculations (dpi). Disease evolution generally halts after 2 weeks post inoculation whereby symptoms could be seen only on older leaves (affected at the time of inoculation). By contrast, new leaves that emerged after 10 dpi remain apparently healthy. After 30 dpi, the entire inoculated plants could appear healthy and the only difference between negative control treatments and those inoculated with the pathogens consist in the growth (reduced growth of plants inoculated with the pathogen). Under field conditions, however, several circumstances could lead to the optimal conditions conducive to continuous bacterial infection (constant availability of the inoculum mostly the secondary one, higher availability of ports of entry, etc.). The contrasting results

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between the field and laboratory experiments could, in part, be due to these reasons. In some cases, higher disease severity of vascular pathogens under field conditions could be due to the infected seeds (seedborne bacteria), presence of pathogen inoculum in the soil (in particular when infected plant debris is buried), and cultivation of plant species in soils infested by nematodes. The latter damage root systems providing a high number of ports of entry for soilborne vascular bacterial pathogens. For example, C. michiganensis on tomato (De Leon et al., 2011); R. solanacearum on tomato, potato, and ginger (Genin, 2010); and X. campestris on cruciferous crops (Vincente and Holub, 2013) cause economically important vascular diseases leading to devastating damage. In such circumstances, all plant parts (leaves, stems, fruits) are systemically affected by the pathogen. Vascular pathogens enter into the water-conducting xylem vessels of the host, proliferate within the vessels, and cause water blockage that leads to the sudden plant wilting. However, the blockage of the xylem vessels may not be necessarily caused by the pathogens themselves but often by the host reactions to invasion. In such cases, the production of gel-like materials could occur, which serves as potential barriers to prevent spread of the pathogens in the vessels. The literature reports suggest that P. syringae mostly causes foliar and not vascular diseases on annual plants (Lamichhane et al., 2015). This calls into question the capacity of P. syringae to systemically spread along the vessels of annual plants, although it should yet be investigated. In contrast, systemic movement of P. syringae in the vascular system of plants is very common in woody plant species (Lamichhane et al., 2014). At this point, one begins to wonder why P. syringae does not cause vascular diseases on herbaceous plants with fragile mechanical structure and does so on woody plants equipped with a robust structure. Which characteristics differentiate woody plants from herbaceous ones? There could be three major reasons: First, woody plants are characterized by a long life cycle (i.e., life span of decades or even centuries) and consequently under field conditions they are constantly exposed to several biotic and abiotic stresses that predispose them to a most severe form of attack. By contrast, annual plants have a very short (4e 5 months) life cycle and thus they are exposed to stress to a lower extent such as mechanical damages. The latter creates the ports of entry that allows bacterial pathogens to access into plant tissues and begin the infection process. Second, because of its different mechanical and anatomical structures as well as long life cycle, woody plants offer an impressive number of entry ports for bacterial pathogens (Lamichhane et al., 2014). Mechanical damage

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caused by natural (hailstones, frost damage) or manmade activities (pruning, grafting) often creates wounds on woody plant tissues, ideal for bacterial pathogens to access. In addition, natural opening such as stomata, hydathodes, and leaf scars that form following leaf shedding in autumn constantly expose woody plants to severe risks. Ubiquitous bacterial pathogens such as P. syringae and saprophytic X. arboricola get access into plant tissues during autumn through thousands of leaf scars created simultaneously following leaf shedding (Lamichhane et al., 2014; Lamichhane, 2014). A large number of leaf scars may be infected simultaneously under field conditions while only one entry port is used during experimental condition (either stomata for leaf inoculation or wound for stab inoculation). This suggests that devastating damages commonly observed under field conditions are due to bacterial infection occurring simultaneously through different entry ports within the same plant. Subsequently, each individual infection point could merge together over time with advancing infections and cause collapse of the whole plant. Both field and laboratory inoculations on systemic migration of P. syringae showed a very limited systemic movement of the pathogen from the inoculated points. The farthest point reached by this pathogen in plant vessels ranged from 3 to 9 cm based on the plant species tested (Roos and Hatting, 1987; Scortichini and Lazzari, 1996; Whitesides and Spotts, 1991).

7. DIFFERENCE IN THE GROWTH STAGE OF PLANT MATERIALS INFECTED One of the reasons for inconsistent results in experimental observations compared to natural infections consist in the type of plant material used for the inoculation. Plant materials used for experimental inoculations are seed-grown (annual plants) or vegetatively propagated (woody plants, in general) potted materials. For woody plants, because field symptoms can be reproduced through artificial infections, it is desirable that adult plants of the same age trained homogeneously are used. However, adult plants are difficult to study, and hence, for experimental purposes young plants have to be used. Overall, woody plants ranging from few months to few (2e3) years old are used for experimental inoculation studies while natural infections occurs on decades-old or even secular plants. As for annual plants, under field conditions, bacterial disease epidemics are very common during the flowering or fruit-bearing stage. The latter represents much advanced phase than those used for the experimental inoculations

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(three to four true-leaf stage). Moreover, the substrates used for potting are sterile and nutrient rich, which markedly differ from that of the soil under field conditions. The literature reports are very heterogeneous in terms of the type of plant material used for the pathogenicity tests. They range from detached plant parts such as buds, fruit, leaves, and twigs (Gilbert et al., 2010; Moragrega et al., 2003; Scortichini et al., 2003) to cotyledons (Cook et al., 1990). However, detached plant parts are deprived of defense system and consequently the level of pathogen aggressiveness observed on such a plant part does not correspond to the pathogenicity test performed in planta. Likewise, plant cotyledons used to measure the aggressiveness of a given pathogen give false-positive results since in this stage plants are deprived of the defense system. Indeed, plants are characterized by age-related (ontogenic) resistance to pathogens (Ficke et al., 2002), although this resistance may differ from one pathosystem to another. Overall, resistance of plant species to bacterial diseases increases with age. Age-related resistance has been reported in P. syringaeearabidopsis (Kus et al., 2002; Wilson et al., 2013), X. campestriserice (Koch and Mew, 1991), Pseudomonas chicoriiedwarf achefflera (Chase and Jones, 1986), C. michiganensisetomato (Chang et al., 1992), and Acidovorax citrulliecucurbitaceae (Bahar and Burdman, 2010) interactions. Another parameter that affects woody plants under field conditions, than those used in the experimental inoculations, is the constant pressure of cultural practices such as grafting, pruning, fertilization, treatments, and irrigation. By contrast, in experimental conditions only irrigation is the practice commonly used. All these itinerary practices influence crop health and its consequent reaction to microorganisms. Factors such as rootstock selection, height of grafting, and early fall pruning affect bacterial disease susceptibility of stone fruits (Prunier et al., 1999; Vigoroux et al., 1997).

8. CONCLUSION A broader vision is needed in plant pathology to understand the complex interactions among host, pathogen and beneficial microflora, and climatic and edaphic factors that are very common under field conditions. These factors not only are responsible for the occurrence or avoidance of a given plant disease but also affect disease severity. Pathogenicity studies in the laboratory and studies on molecular and biochemical signaling

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developed during the pathogenesis are useful but not sufficient to explain field epidemics. Efforts are thus needed to understand the effect of a set of factors on disease occurrence and development, under experimental conditions, rather than focusing on the effect of each individual factor applied one at a time. Simulation studies are needed in experimental conditions to better understand which of these factors combined together lead to a higher risk of disease development with consequent yield losses. The understanding of such complex field phenomena might have an important implication in pest risk analysis. In addition, such information might also help in breeding for resistance given that plant cultivars selected for more stress factors are less likely prone to disease susceptibility compared to plant genotypes selected for a particular biotic stress. Taken together, avoiding stress factors during plant growth appears to be important since most of the bacterial diseases occur when plants are stressed. To this aim, adequate cultivation areas should be selected based on the optimal pedoclimatic conditions that allow plants to produce maximum yield without being stressed.

ACKNOWLEDGMENTS The list of people to be acknowledged would be too long to specify. I thank Claudia Bartoli for critical reading of the manuscript. I am grateful to all my colleagues whom I bothered several times with many questions. Particular thanks to all agronomists, plant pathologists, field technicians, and farmers who shared their practical experiences with me.

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