1.1.1 The ascomycete genus Verticillium
The plant disease Verticillium wilt is caused by members of the soil-borne ascomycete genus Verticillium, which contains ten species: V. albo-atrum sensu stricto, V. alfalfae, V. nonalfalfae, V. dahliae, V. isaacii, V. klebahnii, V. longisporum, V. nubilum, V. tricorpus and V. zaregamisanum (Inderbitzin et al., 2011a; Inderbitzin & Subbarao, 2014). Conidiophores carry verticillate arranged phialides as conidia producing cells, giving the genus the name Verticillium (Pegg & Brady, 2002). These species are distributed in temperate and subtropical regions world-wide and vary in their host ranges (Pegg, 1984; Fradin & Thomma, 2006). The induced symptoms differ between hosts as well as between isolates from a certain species and may comprise stunting, chlorosis, necrosis, early senescence, defoliation, vein clearing, brownish vascular discoloration, or stem striping (Fradin & Thomma, 2006; Depotter et al., 2016).
Verticillium species produce different dormant structures as resting mycelium, chlamydospores, or microsclerotia, which can persist in the soil (Wilhelm, 1955;
Inderbitzin et al., 2011a; Carroll et al., 2018). Increasing temperatures due to climate change are assumed to increase the regional distribution and the economic impact of plant pathogens (Velásquez et al., 2018), like Verticillia, allowing more successful overwintering, extension of the growth season, and potentially earlier infection of younger plants. Furthermore, severity of disease symptoms induced by several Verticillium species was observed to be correlated with increasing temperatures (Koike et al., 1994; Jabnoun-Khiareddine et al., 2006; Siebold & von Tiedemann, 2013).
Available treatments for disease control include planting of resistant varieties, soil fumigation, and crop rotation with non-susceptible plants, but are costly, have questionable effects on the environment, or are not effective (Subbarao et al., 2007;
Klosterman et al., 2009; Enebak et al., 2012; Carroll et al., 2018).
V. dahliae causes vascular wilting disease in almost 200 host species, including a broad range of crop plants and, therefore, has a great economic impact (Pegg & Brady, 2002;
Luo et al., 2014). Even if the species V. dahliae has a broad host range, single isolates of the species show altered aggressiveness on different plant hosts and can even colonize plants without induction of disease symptoms (Resende et al., 1994; Zeise &
von Tiedemann, 2002; Pegg & Brady, 2002; Gibriel et al., 2019). Other members of the genus display narrower host ranges and distributions, however, significant economic losses are caused by V. albo-atrum, V. alfalfae, V. nonalfalfae, V. tricorpus, and
V. zaregamsianum (Inderbitzin & Subbarao, 2014). The species V. isaacii, V. klebahnii, and V. nubilum are pathogens with minor economic impact (Inderbitzin & Subbarao, 2014). V. longisporum is the only interspecific hybrid of the genus with an amphidiploid genome (Ingram, 1968), mainly virulent on Brassicaceae (Zeise & von Tiedemann, 2002;
Eynck et al., 2007). Recently, preliminary data from field trials in the United Kingdom were published, suggesting inconsistent impact on yield reductions caused by rapeseed infections with V. longisporum despite the presence of disease symptoms (Depotter et al., 2019). The two species V. dahliae and V. longisporum are in scope of this study.
1.1.2 Life cycle of Verticillium
Species of the genus Verticillium induce Verticillium wilt, a monocyclic disease with one infection cycle per growing season (Fradin & Thomma, 2006; Klosterman et al., 2011;
Depotter et al., 2016a). V. dahliae and V. longisporum form thick-walled, black melanized microsclerotia from swollen, septate hyphae as a characteristic dormant structure (Griffiths, 1970; Pegg & Brady, 2002; Figure 1, bottom). These resting structures persist in the soil through the winter or, if necessary, for up to 14 years (Wilhelm, 1955).
Recognition of root exudates or plant surface molecules of an appropriate host is the first step in pathogen-plant communication, inducing germination of the fungus (Berlanger &
Powelson, 2000). The fungus initially colonizes the root surface and invades the plant via natural root wounds, or by development of swollen hyphal tips, which were described as penetration structures, named hyphopodia (Pegg & Brady, 2002; Reusche et al., 2014;
Zhao et al., 2014, 2016; Su et al., 2018; Figure 1, left). Root tips, wounds, and lateral root hairs are favored entry points (Fitzell et al., 1980; Eynck et al., 2007; Vallad &
Subbarao, 2008; Su et al., 2018).
From cortical cells hyphae migrate towards the vascular parenchyma and xylem vessels by intercellular growth and some of them successfully reach the xylem (Klosterman et al., 2009). Asexual spores are formed and spread within the vascular system via the transpiration stream (Klosterman et al., 2009). The size and shape of these conidia are the major morphological characteristic to distinguish between V. dahliae and V. longisporum. V. dahliae forms conidia of ovoid shape and a length between 3.5 to 5.5 µm, whereas the name V. longisporum hints to the formation of elongated conidia of 7.1 to 8.8 µm (Karapapa et al., 1997; Collins et al., 2003; Tran et al., 2013; Figure 1, top).
A switch from the biotrophic to the necrotrophic life style corresponds with the colonization of tissues neighboring the xylem (Fradin & Thomma, 2006). Disease symptoms induced by V. dahliae vary between fungal isolates and host plants and
include stunting, chlorosis, necrosis, early senescence, defoliation, vein clearing, and brownish vascular discoloration (Fradin & Thomma, 2006; Figure 1, right).
Figure 1: Verticillium life cycle. V. dahliae and V. longisporum form black melanized microsclerotia as dormant structures. In the presence of a suitable host the fungus germinates and grows into direction of the plant (Berlanger & Powelson, 2000). It colonizes the root surface and invades root tips, lateral root hairs, or natural root wounds by intercellular growth or formation of hyphopodia (Fitzell et al., 1980; Eynck et al., 2007; Vallad
& Subbarao, 2008; Reusche et al., 2014; Zhao et al., 2014, 2016). Hyphae migrate from the cortex to the xylem vessels (Klosterman et al., 2009). Conidia are formed and spread within the vascular system with the transpiration stream (Klosterman et al., 2009). The fungus starts to colonize tissues neighboring the xylem with or without induction of disease symptoms.
Under laboratory conditions the most obvious disease symptoms induced by V. longisporum in rapeseed plants or V. dahliae in tomato plants is stunting. In the dying host or under nutrient-limited conditions the fungus starts microsclerotia formation. Left: electron microscopy picture of V. dahliae growing on Arabidopsis thaliana roots (upper, Rabea Schlüter, Greifswald) and confocal fluorescence microscopy pictures from V. dahliae expressing ectopic GFP colonizing A. thaliana root tips stained in red with propidium iodide/silwet solution (lower). Top: V. dahliae forms smaller conidia (3.5 to 5.5 µm) with ovoid shape, whereas V. longisporum forms elongated conidia (7.1 to 8.8 µm). Right: Rapeseed plants 35 days after root dipping into distilled water or V. longisporum Vl43 spore solution (top). Tomato plants 21 days after root dipping into distilled water or V. dahliae JR2 spore solution (lower). Bottom: Microscopy picture of microsclerotia formed by V. dahliae nine days after spot inoculation of 50 000 spores onto cellulose containing medium (left) and electron microscopy picture of microsclerotia formed by V. dahliae after four days on solid simulated xylem medium (Rabea Schlüter, Greifswald).
V. longisporum-induced symptoms in rapeseed plants in the field are stem striping due to necrosis of the cortical tissue, whereas no wilting can be observed (Heale & Karapapa, 1999; Depotter et al., 2016a). In contrast, artificial infection of rapeseed plants with pathogenic V. longisporum isolates by root dipping of seedlings results in stunting, chlorosis, necrosis, and increased branching (Zeise & von Tiedemann, 2002; Eynck et al., 2007, 2009; Floerl et al., 2008; Depotter et al., 2016a; Figure 1, right). Verticillia are able to colonize plants without induction of disease symptoms. This can be observed either because the host is not susceptible, these plants are commonly considered as
“non-host” or better as “asymptomatic host” plants (Malcolm et al., 2013), or because the Verticillium isolate is “non-pathogenic”.
The fungus recognizes limited nutrient availability in the dying host plant or in plant debris, which induces the formation of resting structures (Fradin & Thomma, 2006).
These resting structures remain in the soil until perception of a new host. A sexual reproduction stage has not been described for any species of the genus (Short et al., 2014).
During all steps of the plant colonization cycle fungi perceive signals from the host environment, which induce differentiated development. On the other hand, the fungus sends signals like effectors to invade and suppress the plant immune system, which is required for successful colonization.