Apple fruits (Malus x domestica) and the pathogen Colletotrichum: Insights in fruit susceptibility changes during a postharvest ripening setting

A major challenge in fruit production nowadays is to limit losses due to postharvest diseases. Despite optimized storage conditions and the application of fungicides, postharvest fungal pathogens are still responsible for considerable losses. The postharvest pathogen Colletotrichum is a recent problem on apple fruits and information is still limited. This pathogen is not visible at harvest due to a quiescent phase. It is only during storage and due to ripening that the pathogen becomes active when fruits become more susceptible and necrotic lesions appear on the fruits. The main objective of our research is to get insights into the specific plant-pathogen interaction Malus x domestica - Colletotrichum. Fruits have passive and active defense to overcome the multiple attacks by fungal pathogens. The focus in this manuscript is mainly towards the passive defense, more specifically the role of the physiological status of fruits and some putative defense metabolites in the susceptibility changes to Colletotrichum. Since Colletotrichum is a relatively new and unknown postharvest pathogen on apple fruit in Belgium (and Europe), we firstly monitored the presence of this pathogen on apple fruits and executed an identification of the different species present by different methods. Briefly we could conclude that the postharvest pathogen Colletotrichum is an important threat for apple fruits in Belgium. Based on sequence analysis of six different genetic regions six different Colletotrichum spp. were identified on 21 different apple cultivars from Belgian orchards: C. fioriniae, probably C. kahawae, C. salicis, C. rhombiforme, C. acutatum and C. godetiae. Colletotrichum fioriniae was found to be the most present and pathogenic species in Belgian orchards. Reliable morphological discrimination between all species, seems not possible. Inoculation assays on two apple cultivars, the more susceptible 'Pinova' and the less susceptible 'Nicoter', revealed a significant difference in pathogenicity among isolates and among Colletotrichum species. The pathogenicity tests indicated that isolates coming from another host species, e.g. strawberry, are also pathogenic on apple fruits. Since Colletotrichum is a postharvest pathogen characterized by a quiescent phase in the apple fruit, we investigated fruit susceptibility of nine apple cultivars at harvest and after storage for Colletotrichum fioriniae, and studied the relationship of the susceptibility with some fruit ripening characteristics. Two artificial inoculation techniques were compared and the wound inoculating method had more advantages compared to the nebulization method. Fruits revealed a higher susceptibility after storage compared to harvest and the nine apple cultivars had clear differences in susceptibility to C. fioriniae. The ripening parameters respiration and Brix had a positive and the firmness a negative correlation with the lesion expansion growth rate of C. fioriniae after artificial wound inoculations of the studied cultivars. Based on these findings, more detailed experiments indicated indeed a link between the ripening of apple fruits and the susceptibility to the postharvest pathogen Colletotrichum. Fruits are ripening during the storage time and the changes being cultivar dependent. Cultivar 'Nicoter' again was the less susceptible cultivar compared to 'Pinova', with each cultivar having their specific ripening characteristics. A stepwise regression pointed out that firmness and ethylene production are key parameters correlating to the lesion expansion growth rate (LEGR) of C. fioriniae on apple fruits. Still, it is not correct to make firm conclusions about physiological fruit changes and susceptibility issues. It is expected that a combination of parameters underlying these physiological changes will make apple fruits more or less susceptible. Besides ripening aspects, the preformed antifungal polyphenols that were studied didn't shown a clear link with the susceptibility for Colletotrichum in apple fruits, while this was often expected in literature. Next to the passive, the induced defense is also very important. In this manuscript a selection of seven induced antifungal polyphenolic compounds and the polyphenol oxidase (PPO) activity was studied on artificial inoculated fruits. The differences in susceptibility, based on symptom expression, were confirmed by qPCR of fungal DNA in the fruit. Next to that, limited significant changes in specific induced polyphenol concentrations in time due to inoculations with C. fioriniae were found. Chlorogenic acid was the most important polyphenol being induced in apple fruits, which is in agreement with previous research on other pathosystems. The induced response based on PPO activity measurements was not visible in our experiments. PPO activity increased in mock wound-inoculated and intact tissue, but not in the pathogen wound-inoculated tissues. For apples that were stored up to 20 weeks, no induction of polyphenols nor significant PPO activities were present, probably due to the fruits being too ripe. We concluded that the induction of polyphenolics and PPO activities alone cannot explain the differences in susceptibility of the cultivars studied. The RNA sequencing analysis executed was exploratory. We focused on the role of ethylene production and cell wall metabolism, the latter related to firmness, since both ripening parameters had a considerable correlation with the changes in fruit susceptibility. The ethylene production measurements during storage and between apple cultivars could partially be explained by the differential expression of transcripts encoding for ACC synthase and ACC oxidase. It is clear that during storage several cell wall degrading enzymes in apple fruits were upregulated and these changes in gene expression, involved in cell wall metabolism, were correlated to the softening of the fruits during storage times and ripening.status: publishe

NOVEL IN VITRO AND IN VIVO MODEL SYSTEMS TO STUDY VAGINAL MICROBICIDE UPTAKE

Novel in vitro and in vivo model systems to study intravaginal microbicide uptakeAnti-HIV microbicides are compounds that are vaginally or rectally administered prior to intercourse to prevent viral transmission. In the search for an effective product, insufficient drug permeation across the vaginal mucosa may cause failed protection in case of microbicides that are active at the level of the HIV host cells, which are mainly localized in the lamina propria. To evaluate the tissue penetration ability of vaginally applied drugs, we established a simple in vitro set up, consisting of an apical and basal chamber that are separated by a layer of HEC-1A cells. This procedure was validated by means of the model microbicides tenofovir, darunavir, saquinavir and dapivirine. The compound s solubility and tissue permeability were identified as key determinants for vaginal uptake. Saquinavir and dapivirine permeation across HEC-1A was low because of poor permeability and solubility, respectively. In contrast, tenofovir showed the highest transport attributable to its good aqueous solubility. In addition, the impact of formulation factors on the permeation potential was investigated. The inclusion of solubilizing cyclodextrins or polyethylene glycol resulted in an enhanced permeation of the hydrophobic compound dapivirine. An excipient-induced reduction in the permeability may however counteract the solubilizing effect, as demonstrated for the emulsion formulation of darunavir. The optimization of drug uptake by inclusion of formulation excipients must take into account this delicate balance. In a next step, the described in vitro approach was applied for the biopharmaceutical assessment of the diaryltriazine compound series which resulted in the selection of UAMC01398 as the lead microbicide candidate, owing to its relatively high aqueous solubility in addition to its beneficial safety and activity profile. Two stable and safe aqueous-based gel formulations were identified for UAMC01398 including a non-solubilizing gel and a gel containing the solubilizer sulfobutyl ether-ß-cyclodextrin (SBE-ßCD, 5%); in addition, a film delivery system, consisting of the excipients hydroxypropylmethylcellulose and polyethylene glycol 400 with UAMC01398 in the amorphous state, was developed. Compared to the non-solubilizing gel, the SBE-ßCD gel and the film formulation increased the UAMC01398 uptake both in vitro across HEC-1A cell layers and in vivo in rabbits. The developed formulations are suitable for the vaginal delivery of UAMC01398 and allow further in vivo evaluation of the microbicide potential of this compound.The possible use of a supersaturation strategy to overcome solubility issues of hydrophobic microbicides was demonstrated for dapivirine. Despite being in a thermodynamically unstable state, supersaturated dapivirine showed not to precipitate in the formulation vehicle as such and in biorelevant fluids in the presence of several excipients including hydroxypropylmethylcellulose, polyethylene glycol 1000 and cyclodextrins. Dapivirine transport across HEC-1A cell layers was higher for supersaturated gels compared to suspension gels. The supersaturated dapivirine (500 µM) gel containing 2.5% of SBE-ßCD significantly increased vaginal drug uptake in rabbits compared to a non-solubilizing suspension gel and an SBE-ßCD suspension gel. The supersaturation approach thus allows the formulation of hydrophobic microbicides at concentrations above their solubility, thereby enhancing their vaginal permeation.Vaginal uptake may also be influenced by drug transporters expressed in the vaginal epithelium. We confirmed the protein expression of the efflux transporters Pgp, BCRP and MRP-2 in endocervical and vaginal tissue of premenopausal women. Several microbicide candidates including darunavir, saquinavir and maraviroc could be categorized as Pgp and MRP-2 substrates and their disposition may thus be affected by these transporters. The Pgp transporter was observed to significantly reduce the vaginal uptake of the model Pgp substrate talinolol in vivo in rabbits when formulated in a neutral, but not in an acidic gel. Consequently, the expression of efflux transporters may limit the vaginal permeation of their substrates, including certain microbicides. In conclusion, we propose the implementation of in vitro solubility and permeability evaluation, including the assessment of transporter interactions, to estimate the vaginal tissue permeation potential of microbicides prior to progressing into animal or clinical studies. This procedure may contribute to the selection of promising microbicide candidates, as illustrated for the diaryltriazines, and to successful formulation development, as shown for UAMC01398 and dapivirine. The in vitro drug transport across HEC-1A cell layers correlated well with the in vivo vaginal permeation as determined in rabbits. Formulation approaches that install solubilization or supersaturation are indicated to enhance vaginal drug uptake of poorly soluble microbicides.status: publishe

Early identification of availability issues for poorly water-soluble microbicide candidates in biorelevant media: a case study with saquinavir

In the search for a successful HIV microbicide, many poorly water-soluble antiviral agents are currently being investigated. Unfortunately, solubility and precipitation issues may limit intravaginal concentrations and thus availability of these agents upon application of an aqueous gel formulation. In the present study, we evaluated the in vitro precipitation behavior of the HIV protease inhibitor saquinavir in vaginal and seminal fluid simulants (VFS and SFS). Despite its limited solubility, the mesylate salt of saquinavir enables formulation of sufficiently high concentrations (2.5 mM, i.e. ca. 10(5)-fold in vitro IC(50) values) in a standard aqueous vehicle. While saquinavir stays in solution upon dilution with VFS, SFS induces precipitation of saquinavir, resulting in a 5-fold reduced availability and antiviral potency. Inclusion of the solubilizing excipients polyethylene glycol 1000 (12%) and hydroxypropyl-β-cyclodextrin (2.5%) was required to avoid saquinavir precipitation in SFS and to restore the antiviral potency of the formulation. This study illustrates the importance of identifying solubility and precipitation issues of microbicide candidates in biorelevant media and provides a simple in vitro procedure to implement this evaluation in early microbicide development.status: publishe

Identification and pathogenicity assessment of Colletotrichum isolates causing bitter rot of apple fruit in Belgium

Worldwide Colletotrichum spp. have been identified as a problem in the apple production. This is the first study executed and confirming the presence of Colletotrichum spp. causing the postharvest disease bitter rot on apple fruits in Belgium. The identification, genetic diversity of Colletotrichum isolates (present in Belgian apple orchards) their morphological traits and pathogenicity on two apple cultivars (cvs. Pinova and Nicoter) with a different level of susceptibility were studied. Based on sequence analysis of six different gene regions beta-tubuline (TUB2), histone H3 (HIS3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 gene (CHS-1), actin (ACT) and the Internal Transcriber Spacer (ITS) gene region, six different Colletotrichum spp., belonging to either the C. acutatum or C. gloeosporioides complexes, were isolated from twenty-one apple cultivars in three Belgian orchards: C. fioriniae, probably C. kahawae, C. salicis, C. rhombiforme, C. acutatum and C. godetiae. Colletotrichum godetiae was found to be the most present and pathogenic species in Belgian orchards. The species C. rhombiforme was found and identified on apple fruit for the first time. Reliable morphological discrimination between species, based on features such as in vitro growth rate, colony colour and spore measurements, is not possible. As such, molecular identification appears to outperform morphological analysis and was in this study the most ideal tool for identifying unknown isolates of Colletotrichum species. Inoculation assays on two apple cultivars revealed a significant difference in pathogenicity among isolates and among Colletotrichum species. The pathogenicity tests also showed that isolates coming from another host species, e.g. strawberry, are also pathogenic on apple fruits. Cultivar Pinova appeared to be more susceptible to bitter rot than cv. Nicoter. Given the difficulties with managing Colletotrichum infections, additional knowledge on the pathogen and the plant-pathogen interaction is essential for effective disease control

Identification and pathogenicity assessment of Colletotrichum isolates causing bitter rot of apple fruit in Belgium

Worldwide Colletotrichum spp. have been identified as a problem in the apple production. This is the first study executed and confirming the presence of Colletotrichum spp. causing the postharvest disease bitter rot on apple fruits in Belgium. The identification, genetic diversity of Colletotrichum isolates (present in Belgian apple orchards) their morphological traits and pathogenicity on two apple cultivars (cvs. Pinova and Nicoter) with a different level of susceptibility were studied. Based on sequence analysis of six different gene regions beta-tubuline (TUB2), histone H3 (HIS3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 gene (CHS-1), actin (ACT) and the Internal Transcriber Spacer (ITS) gene region, six different Colletotrichum spp., belonging to either the C. acutatum or C. gloeosporioides complexes, were isolated from twenty-one apple cultivars in three Belgian orchards: C. fioriniae, probably C. kahawae, C. salicis, C. rhombiforme, C. acutatum and C. godetiae. Colletotrichum godetiae was found to be the most present and pathogenic species in Belgian orchards. The species C. rhombiforme was found and identified on apple fruit for the first time. Reliable morphological discrimination between species, based on features such as in vitro growth rate, colony colour and spore measurements, is not possible. As such, molecular identification appears to outperform morphological analysis and was in this study the most ideal tool for identifying unknown isolates of Colletotrichum species. Inoculation assays on two apple cultivars revealed a significant difference in pathogenicity among isolates and among Colletotrichum species. The pathogenicity tests also showed that isolates coming from another host species, e.g. strawberry, are also pathogenic on apple fruits. Cultivar Pinova appeared to be more susceptible to bitter rot than cv. Nicoter. Given the difficulties with managing Colletotrichum infections, additional knowledge on the pathogen and the plant-pathogen interaction is essential for effective disease control.</p

Identification and pathogenicity assessment of Colletotrichum isolates causing bitter rot of apple fruit in Belgium

© 2018, Koninklijke Nederlandse Planteziektenkundige Vereniging. Worldwide Colletotrichum spp. have been identified as a problem in the apple production. This is the first study executed and confirming the presence of Colletotrichum spp. causing the postharvest disease bitter rot on apple fruits in Belgium. The identification, genetic diversity of Colletotrichum isolates (present in Belgian apple orchards) their morphological traits and pathogenicity on two apple cultivars (cvs. Pinova and Nicoter) with a different level of susceptibility were studied. Based on sequence analysis of six different gene regions beta-tubuline (TUB2), histone H3 (HIS3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 gene (CHS-1), actin (ACT) and the Internal Transcriber Spacer (ITS) gene region, six different Colletotrichum spp., belonging to either the C. acutatum or C. gloeosporioides complexes, were isolated from twenty-one apple cultivars in three Belgian orchards: C. fioriniae, probably C. kahawae, C. salicis, C. rhombiforme, C. acutatum and C. godetiae. Colletotrichum godetiae was found to be the most present and pathogenic species in Belgian orchards. The species C. rhombiforme was found and identified on apple fruit for the first time. Reliable morphological discrimination between species, based on features such as in vitro growth rate, colony colour and spore measurements, is not possible. As such, molecular identification appears to outperform morphological analysis and was in this study the most ideal tool for identifying unknown isolates of Colletotrichum species. Inoculation assays on two apple cultivars revealed a significant difference in pathogenicity among isolates and among Colletotrichum species. The pathogenicity tests also showed that isolates coming from another host species, e.g. strawberry, are also pathogenic on apple fruits. Cultivar Pinova appeared to be more susceptible to bitter rot than cv. Nicoter. Given the difficulties with managing Colletotrichum infections, additional knowledge on the pathogen and the plant-pathogen interaction is essential for effective disease control.status: publishe

Vaginal expression of efflux transporters and the potential impact on the disposition of microbicides in vitro and in rabbits

In order to reach sufficiently high tissue concentrations and thus be effective, vaginally applied anti-HIV microbicides that are active at the level of the immune cells must permeate across the cervicovaginal mucosal layer. Cellular efflux transporters, such as Pgp, BCRP, and MRP-2, have been demonstrated to greatly affect drug disposition at different sites in the body including the intestine and the blood-brain barrier; their possible role on drug uptake from the female genital tract, however, has not been elucidated yet. In the present study, the protein expression of Pgp, BCRP, and MRP-2 in endocervical and vaginal tissue of premenopausal women was confirmed by Western blot analysis. To enable the assessment of transporter effects in vitro, the identification of an appropriate cervicovaginal cell line was pursued. The cervical SiHa cell line was observed to express mRNA of the 3 studied transporters, but only MRP-2 was found to be active. Consequently, the established Caco-2 cell line was utilized as an alternative in which the interaction of 10 microbicide candidates with the efflux transporters was studied. Darunavir, saquinavir, and maraviroc were identified as Pgp and MRP-2 substrates. The impact of Pgp on in vivo drug disposition was further examined for the model Pgp substrate talinolol in rabbits. Its vaginal uptake was significantly reduced by Pgp-mediated efflux when formulated in a neutral but not in an acidic gel. Our findings indicate the expression of a functional Pgp transporter in the vaginal mucosa that may severely reduce the vaginal uptake of Pgp substrates, including certain microbicide candidates, especially in women with an increased vaginal pH