The Biochemical Basis of Pathogenicity and Host-Specificity of Penicillium Digitatum on Citrus

In this work we report that volatiles emitted from wounded citrus peel play a major role in host recognition by Penicillium digitatum. Volatiles of various citrus cultivars had a pronounced stimulatory effect on germination and germ tube elongation of green mold pathogen. When exposed to volatiles from grapefruit, the percentage of P. digitatum spores germinated on minimal media was 10-fold higher as compared to the control. In contrast, conidia germination and growth in non-host pathogens, Botrytis cinerea and Penicillium expansum, were either not affected or inhibited by the citrus peel volatiles. A GS-MS analysis of volatile compounds in the wound head space of various citrus fruit cultivars revealed that limonene is the major compound, suggesting it as a potential chemical regulator of germination in P. digitatum. After reaching the wound, to successfully colonize the entire fruit, P. digitatum needs to overcome defense mechanisms in the host tissue. Indeed, we found that P. digitatum actively suppresses a defense-related hydrogen peroxide (H2O2) burst in the citrus peel. In contrast, inoculation of citrus fruit with a non-pathogenic fungus, Penicillium expansum, triggers a massive production of H2O2 in host cells. Important, that initially (8 to 17h after inoculation) both fungi trigger an elevation in H2O2 levels in lemon peel disks. Later, approximately 25h after inoculation P. digitatum succeeds to significantly suppress H2O2 production by host cells, while in discs inoculated with P. expansum, the level of H2O2 was 2.5-fold above the control value at this time point. Suppression of H2O2 production in host tissue by exogenous citric acid significantly (P≤0.05) enhanced pathogenicity of P. digitatum and even allowed a non-pathogenic P. expansum to develop large lesions on lemons, oranges and grapefruits. These results, together with recent reports suggesting the potential involvement of citric acid in green mold pathogenesis, indicate that the ability to suppress hydrogen peroxide production in host tissue plays an important role in pathogenicity of P. digitatum on citrus fruit. INTRODUCTION Green mold caused by Penicillium digitatum is the most damaging postharvest diseases of citrus fruit. This Penicillium species is specific to citrus fruit and does not cause progressive decay in any other fresh fruit or vegetable crops. While the etiology of P. digitatum is well understood, the physiological and biochemical basis of its narrow host specificity is less clear. Unlike the spores of many fungi, conidia of P. digitatum do not germinate on a medium lacking a carbon source such as water agar or on the surfaces of intact citrus fruit (Kavanagh and Wood, 1971; Eckert et al., 1984), but do germinate if exposed to volatiles released in injured citrus fruit. The mechanism of perception and germination following exposure to stimulatory volatiles released from wounds are not well understood either. Knowledge of this phenomenon is limited to few conflicting reports in which different mixtures of a variety of volatiles were suggested to be responsible for stimulation or inhibition of spore germination in P. digitatum (Davis and Smoot, 1972; Eckert et al., 1984, 1992; French et al., 1978; Eckert and Ratnayake, 1994; 1663 Proc. 6 th International Postharvest Symposium Eds.: M. Erkan and U. Aksoy Acta Hort. 877, ISHS 2010 Rodov et al., 1995). Following germination, and before colonization of fruit tissue, P. digitatum needs to overcome defense mechanisms in the peel. Numerous reports documented the existence of versatile inducible and innate mechanisms of pathogen defense in citrus fruit (Fajardo et al., 1998; Afek et al., 1999; Porat et al., 2003; Hasdai et al., 2004). The majority of studies suggested that flavedo tissue has the highest defense potential (Ballester et al., 2006). In contrast, very little is known about Penicillium-fruit interaction from the pathogen’s perspective. There are few evidences that P. digitatum has the ability to suppress fruit defenses. Ismail and Brown (1979) reported that elevation of phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) activity is inhibited in citrus peel around wounds inoculated with the fungus. Lisker et al. (1983) later demonstrated that PAL activity is also suppressed in areas distant from inoculation sites. More recently, Prusky et al. (2004) suggested that P. digitatum compromises citrus fruit defense by acidifying host tissue, hypothesizing that low ambient pH is optimal for expression and functioning of certain fungal polygalactouronase. However, these findings still do not explain how the entire defense machinery of the citrus fruit (phytoalexins, pathogenesisrelated (PR) proteins, lignification, etc.) is not triggered in response to invasion by P. digitatum or how the fungus disrupts defense signaling pathways in a host in regions distant from the site of inoculation. To identify host-specific signals recognized by green mold pathogen, we proposed to characterize the composition of wound headspace and citrus oil volatiles of three citrus cultivars using GC-MS and to investigate the effect of individual wound volatiles found in wound headspace on spore germination and germ tube elongation of P. digitatum as compared to their effects on two other common postharvest pathogens, P. expansum and Botrytis cinerea, that are not pathogenic on citrus fruit. In addition, to get new insight into mechanisms by which P. digitatum overcomes citrus fruit defenses we investigated whether or not P. digitatum can suppress the production of ROS in host tissue. In doing so we examined the production of H2O2 in citrus fruit after inoculation with a compatible (P. digitatum) and a versatile non-host pathogen (P. expansum). Also of importance was to explore the effect of certain organic acids, suggested to be secreted by P. digitatum (Prusky et al., 2004) on its virulence on citrus fruit. MATERIALS AND METHODS Materials ‘Star Ruby’ red grapefruit (Citrus paradise Macf), ‘Valencia’ oranges (Citrus sinesis), clemetine (Citrus reticulata L.) and lemon (Citrus limon L.) of full harvest maturity were obtained from a nearby orchard. Pear, apple, tomato, pepper, strawberry and avocado fruits were purchased from a local market. Chemicals were of analytical grade and were purchased from Sigma-Aldrich, except 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-DA) purchased from Molecular Probes Invitrogen, Eugene, OR, USA. Fungal Cultures and Preparation of the Inoculum Strains of Penicillium digitatum, Penicillium expansum and Botrytis cinerea, were obtained from the culture collection of the Dept. of Postharvest science, ARO, the Volcani Center. Spore suspensions were prepared from 2to 3-week-old PDA (Potato dextrose Agar, Difco, Detroit, MI) cultures of the pathogens. Conidia were harvested from 2to 3-week-old cultures and final spore concentrations in water were adjusted with a haemocytometer. Detection of Volatiles in the Headspace of Injured Citrus Fruit Collection and detection of volatile compounds present in the wound headspace of different citrus fruits was performed as described by Droby et al. (2008).