Purification and Biochemical Characterization of Polygalacturonase Produced by Penicillium expansum During Postharvest Decay of 'Anjou' Pear

A polygalacturonase (PG) was extracted and purified from decayed tissue of 'Anjou' pear fruit inoculated with Penicillium expansum. Ammonium sulfate precipitation, gel filtration, and cation exchange chromatography were used to purify the enzyme. Both chromatographic methods revealed a single peak corresponding to PG activity. PG enzyme activity from healthy and wounded pear tissue was undetectable, which supports the claim that the purified PG is of fungal origin. The purified enzyme had a molecular mass of 41 kDa and a pI of 7.8. Activity of the PG was not associated with a glycosylated protein. The enzyme was active over a broad pH range from 3 to 6, with optimal activity at 4.5 in sodium citrate and sodium acetate buffers. The optimal temperature for activity was 37 degrees C but the enzyme was also active at 0, 5, 10, 20, and 50 degrees C. Thin-layer chromatographic analysis of PG hydrolysis products showed that the enzyme exhibits endo- and exo-activity. The purified enzyme macerated tissue in vitro causing approximate to 30% reduction in mass of pear plugs compared with approximate to 17% reduction for apple. Additionally, it produced 1.5-fold more soluble polyuronides on pear than apple tissue. This work shows for the first time the production of a PG by P. expansum during postharvest decay of pear fruit is different from the previously described PG produced in decayed apple fruit by the same pathogen

Incidence, Speciation, and Morpho-Genetic Diversity of Penicillium spp. Causing Blue Mold of Stored Pome Fruits in Serbia

Blue mold, caused by Penicillium spp., is one of the most economically important postharvest diseases of pome fruits, globally. Pome fruits, in particular apple, is the most widely grown pome fruit in Serbia, and the distribution of Penicillium spp. responsible for postharvest decay is unknown. A two-year survey was conducted in 2014 and 2015, where four pome fruits (apple, pear, quince, and medlar) with blue mold symptoms were collected from 20 storage locations throughout Serbia. Detailed morphological characterization, analysis of virulence in three apple cultivars, and multilocus phylogeny revealed three main Penicillium spp. in order of abundance: P. expansum, P. crustosum, and P. solitum. Interestingly, P. expansum split into two distinct clades with strong statistical support that coincided with several morphological observations. Findings from this study are significant and showed previously undocumented diversity in blue mold fungi responsible for postharvest decay including the first finding of P. crustosum, and P. solitum as postharvest pathogens of quince and P. crustosum of medlar fruit in the world, and P. expansum of quince in Serbia. Data from this study provide timely information regarding phenotypic, morphological and genotypic plasticity in P. expansum that will impact the design of species-specific detection tools and guide the development of blue mold management strategies

Exposure in vitro to an Environmentally Isolated Strain TC09 of Cladosporium sphaerospermum Triggers Plant Growth Promotion, Early Flowering, and Fruit Yield Increase

A growing number of bacteria and fungi have been found to promote plant growth through mutualistic interactions involving elements such as volatile organic compounds (VOCs). Here, we report the identification of an environmentally isolated strain of Cladosporium sphaerospermum (herein named TC09), that substantially enhances plant growth after exposure in vitro beyond what has previously been reported. When cultured on Murashige and Skoog (MS) medium under in vitro conditions, tobacco seedlings (Nicotiana tabacum) exposed to TC09 cultures for 20 days increased stem height and whole plant biomass up to 25- and 15-fold, respectively, over controls without exposure. TC09-mediated growth promotion required >5 g/L sucrose in the plant culture medium and was influenced by the duration of exposure ranging from one to 10 days, beyond which no differences were detected. When transplanted to soil under greenhouse conditions, TC09-exposed tobacco plants retained higher rates of growth. Comparative transcriptome analyses using tobacco seedlings exposed to TC09 for 10 days uncovered differentially expressed genes (DEGs) associated with diverse biological processes including cell expansion and cell cycle, photosynthesis, phytohormone homeostasis and defense responses. To test the potential efficacy of TC09-mediated growth promotion on agricultural productivity, pepper plants (Capsicum annuum L.) of two different varieties, Cayenne and Minisweet, were pre-exposed to TC09 and planted in the greenhouse to monitor growth, flowering, and fruit production. Results showed that treated pepper plants flowered 20 days earlier and yielded up to 213% more fruit than untreated controls. Altogether the data suggest that exposure of young plants to C. sphaerospermum produced VOCs may provide a useful tool to improve crop productivity

Whole-genome comparisons of Penicillium spp. reveals secondary metabolic gene clusters and candidate genes associated with fungal aggressiveness during apple fruit decay

Blue mold is a postharvest rot of pomaceous fruits caused by Penicillium expansum and a number of other Penicillium species. The genome of the highly aggressive P. expansum strain R19 was re-sequenced and analyzed together with the genome of the less aggressive P. solitum strain RS1. Whole genome scale similarities and differences were examined. A phylogenetic analysis of P. expansum, P. solitum, and several closely related Penicillium species revealed that the two pathogens isolated from decayed apple with blue mold symptoms are not each other’s closest relatives. Among a total of 10,560 and 10,672 protein coding sequences respectively, a comparative genomics analysis revealed 41 genes in P. expansum R19 and 43 genes in P. solitum RS1 that are unique to these two species. These genes may be associated with pome fruit–fungal interactions, subsequent decay processes, and mycotoxin accumulation. An intact patulin gene cluster consisting of 15 biosynthetic genes was identified in the patulin producing P. expansum strain R19, while only a remnant, seven-gene cluster was identified in the patulin-deficient P. solitum strain. However, P. solitum contained a large number of additional secondary metabolite gene clusters, indicating that this species has the potential capacity to produce an array of known as well as not-yet-identified products of possible toxicological or biotechnological interest

Distribution and Characterization of Monilinia spp. Causing Apple Fruit Decay in Serbia

Brown rot, caused by Monilinia spp., is an economically important pre and postharvest disease of pome and stone fruits worldwide. In Serbia, apple is the most widely grown pome fruit, and the distribution of economically important Monilinia spp. responsible for apple brown rot is unknown. Hence, we conducted a three year survey, from 2010 to 2012, where 349 isolates were obtained from six orchards and four storage facilities from five different apple cultivars with brown rot symptoms. Morphological characterization of the isolates, multiplex PCR, and phylogenetic analysis revealed four species: M. fructigena, M. laxa, M. fructicola, and Monilia polystroma. All species were found in the orchard and in storage, with M. fructigena predominating, followed by M. polystroma. Representative isolates were analyzed in vitro and in vivo where differences in growth rate, sporulation, and virulence on apple fruit were observed. Findings from this investigation demonstrate diversity in the species responsible for pre and postharvest apple brown rot, which has significant implications for pathogen detection and for developing disease-specific management strategies

The dual nature of Lambertella corni-maris as an apple fruit pathogen and antagonist of Monilinia spp

Lambertella corni-maris was isolated from a dark brown lesion on apple fruit cv. Golden Delicious collected from an orchard, and from stored apple fruit cv. Idared previously colonized with Monilia polystroma. The contrasting origin of the isolates led us to hypothesize that this fungus behaves differently in various contexts, which triggered the investigation of L. corni-maris as a pathogen and antagonist. Following identification based on cultural and molecular characteristics, isolates of different origin were characterized and their cultural, ecological, pathogenic, and antagonistic features were compared. This study has shown that L. corni-maris, originally isolated as an apple fruit pathogen and one as an antagonist of M. polystroma, have similar features in vitro and in vivo. The isolates vary widely in growth rate, cultural morphology, crystal production, intensity of dark zone and co-antagonism with two Monilinia species but can also function as apple fruit pathogens and inhibit and replace different Monilinia spp. on apple fruit. These findings reinforce their dual nature in different host-pathogen interactions. The data from this study are biologically intriguing and practically relevant, and have translative potential to exploit L. corni-maris as a model system for developing strategies to block brown rot of apple and to pinpoint virulence factors in the fungus for yellow rot management

Genomic Resources of Four Colletotrichum Species (C. fioriniae, C. chrysophilum, C. noveboracense, and C. nupharicola) Threatening Commercial Apple Production in the Eastern United States

The genus Colletotrichum includes nine major clades with 252 species and 15 major phylogenetic lineages, also known as species complexes. Colletotrichum spp. are one of the top fungal plant pathogens causing anthracnose and pre- and postharvest fruit rots worldwide. Apple orchards are imperiled by devastating losses from apple bitter rot, ranging from 24 to 98%, which is a serious disease caused by several Colletotrichum species. Bitter rot is also a major postharvest rot disease, with C. fioriniae causing from 2 to 14% of unmarketable fruit in commercial apple storages. Dominant species causing apple bitter rot in the Mid-Atlantic United States are C. fioriniae from the Colletotrichum acutatum species complex and C. chrysophilum and C. noveboracense from the C. gloeosporioides species complex (CGSC). C. fioriniae is the dominant species causing apple bitter rot in the Northeastern and Mid-Atlantic states. C. chrysophilum was first identified on banana and cashew but has been recently found as the second most dominant species causing apple bitter rot in the Mid-Atlantic. As the third most dominant pathogen, C. noveboracense MB 836581 was identified as a novel species in the CGSC, causing apple bitter rot in the Mid-Atlantic. C. nupharicola is a sister group to C. fructicola and C. noveboracense, also causing bitter rot on apple. We deliver the resources of 10 new genomes, including two isolates of C. fioriniae, three isolates of C. chrysophilum, three isolates of C. noveboracense, and two isolates of C. nupharicola collected from apple fruit, yellow waterlily, and Juglans nigra. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

Mining the <i>Penicillium expansum</i> Genome for Virulence Genes: A Functional-Based Approach to Discover Novel Loci Mediating Blue Mold Decay of Apple Fruit

Blue mold, a postharvest disease of pome fruits, is caused by the filamentous fungus Penicillium expansum. In addition to the economic losses caused by P. expansum, food safety can be compromised, as this pathogen is mycotoxigenic. In this study, forward and reverse genetic approaches were used to identify genes involved in blue mold infection in apple fruits. For this, we generated a random T-DNA insertional mutant library. A total of 448 transformants were generated and screened for the reduced decay phenotype on apples. Of these mutants, six (T-193, T-275, T-434, T-588, T-625, and T-711) were selected for continued studies and five unique genes were identified of interest. In addition, two deletion mutants (Δt-625 and Δt-588) and a knockdown strain (t-434KD) were generated for three loci. Data show that the ∆t-588 mutant phenocopied the T-DNA insertion mutant and had virulence penalties during apple fruit decay. We hypothesize that this locus encodes a glyoxalase due to bioinformatic predictions, thus contributing to reduced colony diameter when grown in methylglyoxal (MG). This work presents novel members of signaling networks and additional genetic factors that regulate fungal virulence in the blue mold fungus during apple fruit decay