Botrytis cinerea (Punja and Ni, 2021)

Mahmoud, Mohannad, BenRejeb, Ines, Punja, Zamir K., Buirs, Liam & Jabaji, Suha, 2023, Understanding bud rot development, caused by Botrytis cinerea, on cannabis (Cannabis sativa L.) plants grown under greenhouse conditions, Botany 101 (7), pp. 200-231 : 202-204

Pathogenesis of Botrytis cinerea

Germination process

The interaction between Botrytis cinerea and the host begins with attachment of spores to the plant surface, followed by spore germination, formation of appressorium-like structures or infection cushions (ICs), penetration through the cuticle, host colonization, and finally degradation of host cells ( Fig. 6). Several external conditions affect Botrytis cinerea spore germination, including temperature, humidity, presence of exogenous carbohydrates and nutrients, surface hydrophobicity, and surface hardness ( Fig. 6). These factors can activate signaling pathways in the conidia by interacting with specific protein receptors ( Beris et al. 2021). The attachment process of the spores to the plant surface initially involves hydration of the conidia, which relies on a weak hydrophobic attachment to the host surface ( Doss et al. 1993). A few hours later, germination of conidia initiates a stronger conidia–host attachment ( Doss et al. 1995). The tip of the germ tube branches and swells, forming appressoria-like structure (ALS) or an IC ( Fig. 6) ( Emmett and Parbery 1975; van Kan 2003). The later conidia-adhesion stage is supported by an ECM (extracellular matrix)film, which is a mix of compounds secreted by both germ tubes and later by the IC ( Doss et al. 1995).

Early infection process

Once established, the IC uses mechanical (pressure) and/or enzymatic activity (chemicals) to penetrate the plant cell wall ( Doss 1999; Choquer et al. 2021). The accumulation of specific reactive oxygen species (ROSs), such as hydrogen peroxide (H 2 O 2), during the early infection stages supports the role of these compounds in pathogenesis and the infection process by Botrytis cinerea ( Siegmund and Viefhues 2016) . A number of organic acids, such as citric, succinic, and oxalic acids, are also secreted by Botrytis cinerea , which contribute to the pH adjustment during colonization of host tissues ( Verhoeff et al. 1988; Manteau et al. 2003). Mycotoxins represent another aspect of the pathogenesis arsenal of Botrytis cinerea . Botryanes ( Collado et al. 2000) and botcinins ( Tani et al. 2005, 2006) represent the most studied group of toxins. Botrydial and botcinic acid are common nonselective host phytotoxins that play a role in pathogenesis and necrotrophic activity ( Rebordinos et al. 1996; Dalmais et al. 2011). The roles of these pathogen-derived products in pathogenesis of Botrytis cinerea on cannabis tissues have not been investigated but they are likely to be involved.

Late infection process

Although Botrytis cinerea is typically considered a necrotrophic pathogen that obtains nutrients from dead plant cells, the early infection stage may suppress host cell death to enable successful colonization (Rajarammohan

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2021). In this initial biotrophic stage, in which host cells are not killed, the pathogen may produce various cell death suppressors to permit colonization of the host before activating its kill machinery ( Van Kan et al. 2014; Rajarammohan 2021). Once the pathogen has entered into host tissues, either by direct penetration or through wounds, further development is achieved through secretion of lytic enzymes to breach the plant cell walls, followed by release of phytotoxic metabolites, organic acids, and ROSs that trigger an oxidative burst and programmed cell death (PCD), causing host cell death ( van Kan 2006; Frias et al. 2011). This can occur during plant development and also as a postharvest infection that is common on crops such as strawberries ( El Oirdi et al. 2011; Petrasch et al. 2019).

Latent infection

There is evidence that Botrytis cinerea can also grow in the host as an endophyte (nonsymptomatic) ( Barnes and Shaw 2003). For example, Sowley et al. (2010) reported that systemic spread of Botrytis cinerea occurs in healthy lettuce plants, confirming the endophytic lifestyle of this pathogen. Using an immunolabeling technique, they identified Botrytis cinerea infection in asymptomatic lettuce ( Lactuca sativa ) plants grown indoors. Such infections may represent an association between growing mycelium and host tissues over several weeks/months. This lifestyle may change at a later stage, notably at flowering time, to become pathogenic. In Arabidopsis ( Arabidopsis thaliana ), the pathogen is known to exist in a latent form ( Shaw et al. 2016; Emmanuel et al. 2018). In many cultivated hosts, such as hybrid commercial Primula , lettuce, grapevine, strawberry, kiwifruit, Botrytis cinerea resumes growth when host tissues mature or begin to senesce ( Braun and Sutton 1988; Nair et al. 1995; Michailides and Elmer 2000; Barnes and Shaw 2002, 2003; Sowley et al. 2010). Moreover, quiescent infections of Botrytis cinerea at early stages of plant development, before visible symptoms appear, have been described ( Williamson et al. 2007; Veloso and van Kan 2018). This latent infection was also noted in grapevines, after Botrytis cinerea spores were inoculated into the flower at full bloom in both pot and field trials ( Keller et al. 2003). Interestingly, Botrytis cinerea strains recovered from symptomless lettuce were able to cause lesions on detached leaf pieces ( Sowley et al. 2010). This switching from the latent to pathogenic behavior may be triggered by the surrounding environment, such as elevated stress, moisture, or nutrient limitation ( Barnes and Shaw 2002; Sowley et al. 2010), or when the infected tissue develops to a different growth stage, such as at maturity or during senescence ( Shaw et al. 2016). For example, Botrytis cinerea can remain quiescent for 5–6 weeks in some berry crops, such as raspberries and black currants. In parallel with the increase of the sugar content near harvest, the pathogen begins invading the fruits and causing damage ( Jarvis 1977; Gossen et al. 2014). Whether or not latency or quiescent infections occur in cannabis inflorescences remains to be determined but it is likely taking place. This would represent an important component of the disease cycle and complicate efforts for disease management.

Potential infection sites

At present, the initial infection sites for Botrytis cinerea development on cannabis tissues leading to bud rot have not been confirmed through experimentation. The exposed surface of the stigmas, bracts, inflorescence leaves, or trichome glands could allow for spore attachment and infection. We hypothesize that stigmas are a predominant infection route. Williamson et al. (2007) suggested that the stigmatic fluid on the stigmas on some plants can serve as a nutrient source for conidia and described how Botrytis cinerea hyphae could be observed growing down the style to the ovules in raspberry and strawberry fruits; this follows the same route taken by pollen tubes. On young developing cannabis flowers, for example, the stigmatic surfaces are well exposed during the first few weeks of flower development ( Figs. 7A and 7B). In addition, environmental stresses and maturation of the flowers can cause stigmatic tissues to senesce, potentially providing infection sites on necrotic tissues. On grape flowers, infection of the receptacle area led to more significant disease on berries compared with the stigmas ( Keller et al. 2003).

According to Punja and Ni (2021), inflorescence leaves may also be a point of origin for Botrytis cinerea infection, as they surround the inflorescence, creating a humid microclimate that would favor germination of any spores deposited there ( Figs. 7C and 7D). Early infections are generally seen to originate from the base of these inflorescence leaves ( Fig. 8A). A comparison of four cannabis genotypes that differed in the number of inflorescence leaves formed on the inflorescences showed a positive trend between leaf numbers and enhanced susceptibility to Botrytis cinerea ( Figs. 8B and 8C). These observations are supported by the previous finding that larger, more dense inflorescence structures with more inflorescence leaves ( Fig. 4) are more susceptible to infection. Comparison of total phenolic levels in the foliage leaves (L), inflorescence leaves (S), and inflorescence tissues (F) of four cannabis genotypes showed no correlation with susceptibility. The inflorescence tissues showed higher phenolic levels and yet were found to have the highest susceptibility to Botrytis cinerea ( Fig. 8D). Therefore, the biochemical basis for any observed differences among cannabis genotypes in response to Botrytis cinerea infection remains unknown. The role of the glandular trichomes, which are produced in abundance on inflorescence leaves and bracts of cannabis inflorescences ( Figs. 7E and 7F), in facilitating infection by Botrytis cinerea remains unexplored.