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

Avirulent Isolates of <i>Penicillium chrysogenum</i> to Control the Blue Mold of Apple Caused by <i>P. expansum</i>

Blue mold is an economically significant postharvest disease of pome fruit that is primarily caused by Penicillium expansum. To manage this disease and sustain product quality, novel decay intervention strategies are needed that also maintain long-term efficacy. Biocontrol organisms and natural products are promising tools for managing postharvest diseases. Here, two Penicillium chrysogenum isolates, 404 and 413, were investigated as potential biocontrol agents against P. expansum in apple. Notably, 404 and 413 were non-pathogenic in apple, yet they grew vigorously in vitro when compared to the highly aggressive P. expansum R19 and Pe21 isolates. Whole-genome sequencing and species-specific barcoding identified both strains as P. chrysogenum. Each P. chrysogenum strain was inoculated in apple with the subsequent co-inoculation of R19 or Pe21 simultaneously, 3, or 7 days after prior inoculation with 404 or 413. The co-inoculation of these isolates showed reduced decay incidence and severity, with the most significant reduction from the longer establishment of P. chrysogenum. In vitro growth showed no antagonism between species, further suggesting competitive niche colonization as the mode of action for decay reduction. Both P. chrysogenum isolates had incomplete patulin gene clusters but tolerated patulin treatment. Finally, hygromycin resistance was observed for both P. chrysogenum isolates, yet they are not multiresistant to apple postharvest fungicides. Overall, we demonstrate the translative potential of P. chrysogenum to serve as an effective biocontrol agent against blue mold decay in apples, pending practical optimization and formulation

Assessment of Genetic Diversity of Sweet Potato in Puerto Rico

<div><p>Sweet potato (<i>Ipomoea batatas</i> L.) is the seventh most important food crop due to its distinct advantages, such as adaptability to different environmental conditions and high nutritional value. Assessing the genetic diversity of this important crop is necessary due to the constant increase of demand for food and the need for conservation of agricultural and genetic resources. In Puerto Rico (PR), the genetic diversity of sweet potato has been poorly understood, although it has been part of the diet since Pre-Columbus time. Thus, 137 landraces from different localities around PR were collected and subjected to a genetic diversity analysis using 23 SSR-markers. In addition, 8 accessions from a collection grown in Gurabo, PR at the Agricultural Experimental Station (GAES), 10 US commercial cultivars and 12 Puerto Rican accessions from the USDA repository collection were included in this assessment. The results of the analysis of the 23 loci showed 255 alleles in the 167 samples. Observed heterozygosity was high across populations (0.71) while measurements of total heterozygosity revealed a large genetic diversity throughout the population and within populations. UPGMA clustering method revealed two main clusters. Cluster 1 contained 12 PR accessions from the USDA repository collection, while cluster 2 consisted of PR landraces, US commercial cultivars and the PR accessions from GAES. Population structure analysis grouped PR landraces in five groups including four US commercial cultivars. Our study shows the presence of a high level of genetic diversity of sweet potato across PR which can be related to the genetic makeup of sweet potato, human intervention and out-crossing nature of the plant. The history of domestication and dispersal of sweet potato in the Caribbean and the high levels of genetic diversity found through this study makes sweet potato an invaluable resource that needs to be protected and further studied.</p></div

Twenty-three SSR marker primers with their respective sequence, annealing temperatures, repeat motifs and allele size.

<p>H_O: Observed heterozygosity.</p><p>H_T: Total heterozygosity.</p><p>SSR Source: ^a Buteler <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116184#pone.0116184-Buteler1" target="_blank">[12]</a>; ^b Tseng <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116184#pone.0116184-Tseng1" target="_blank">[15]</a>; ^c Yañez <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116184#pone.0116184-Yaez1" target="_blank">[18]</a>; ^d Benavides (unpublished data; 2002–2003 at CIP); ^e Solis <i>et al.</i> (unpublished data; 2005–2006 developed at CIP); ^f Yada <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116184#pone.0116184-Yada1" target="_blank">[17]</a>.</p><p>Twenty-three SSR marker primers with their respective sequence, annealing temperatures, repeat motifs and allele size.</p

Polymorphic Index Content (PIC) value of the 23 SSR loci analyzed in 167 accessions of sweet potato (<i>Ipomoea batatas</i>).

<p>Polymorphic Index Content (PIC) value of the 23 SSR loci analyzed in 167 accessions of sweet potato (<i>Ipomoea batatas</i>).</p

Summary statistics of genetic diversity estimators at 23 SSR loci for 167 sweet potato samples.

<p>(NoA: Number of Alleles, H_o: Observed Heterozygosity, H_T: Total Heterozygosity, G_is: Inbreeding Coefficient).</p><p>Summary statistics of genetic diversity estimators at 23 SSR loci for 167 sweet potato samples.</p

Delta K values with respect to K (number of groups) according to the calculation method by Evanno <i>et al</i>. [26].

<p>These results were obtained based on the analysis of 23 SSR markers in 167 accessions of sweet potato from the agricultural experimental station in Gurabo, Puerto Rico (12 accessions), the plant genetic resources conservation unit in Griffin, GA (22 accessions) and 137 Puerto Rico landraces.</p

Distribution of the number of alleles per locus analyzed for 23 SSR loci in 167 accessions of sweet potato (<i>Ipomoea batatas</i>).

<p>Distribution of the number of alleles per locus analyzed for 23 SSR loci in 167 accessions of sweet potato (<i>Ipomoea batatas</i>).</p

Population structure and UPGMA clustering based on Euclidean distances of 167 accessions of sweet potato (<i>Ipomoea batatas</i>) analyzed with 23 SSR markers.

<p>Eight accessions are from the agricultural experimental station in Gurabo, Puerto Rico (GAES), 22 from the plant genetic resources conservation unit (PGRCU) in Griffin, GA (12 PR accessions and 10 known US commercial cultivars) and 137 Puerto Rico landraces across the island were analyzed. The eight groups (Group 1: olive green, Group 2: pink, Group: navy blue, Group: orange, Group: purple, Group: lime green, Group: red, and Group: blue) were determined based on STRUCTURE and Evanno <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116184#pone.0116184-Evanno1" target="_blank">[26]</a> analysis. The colors of the branches in the dendrogram also indicates the groups while the highlight of the name refers to PRGCU (olive green), GAES (pink), US cultivars (navy blue) and PR landraces (light gray).</p

The KdmB-EcoA-RpdA-SntB chromatin complex binds regulatory genes and coordinates fungal development with mycotoxin synthesis

Chromatin complexes control a vast number of epigenetic developmental processes. Filamentous fungi present an important clade of microbes with poor understanding of underlying epigenetic mechanisms. Here, we describe a chromatin binding complex in the fungus Aspergillus nidulans composing of a H3K4 histone demethylase KdmB, a cohesin acetyltransferase (EcoA), a histone deacetylase (RpdA) and a histone reader/E3 ligase protein (SntB). In vitro and in vivo evidence demonstrate that this KERS complex is assembled from the EcoA-KdmB and SntB-RpdA heterodimers. KdmB and SntB play opposing roles in regulating the cellular levels and stability of EcoA, as KdmB prevents SntB-mediated degradation of EcoA. The KERS complex is recruited to transcription initiation start sites at active core promoters exerting promoter-specific transcriptional effects. Interestingly, deletion of any one of the KERS subunits results in a common negative effect on morphogenesis and production of secondary metabolites, molecules important for niche securement in filamentous fungi. Consequently, the entire mycotoxin sterigmatocystin gene cluster is downregulated and asexual development is reduced in the four KERS mutants. The elucidation of the recruitment of epigenetic regulators to chromatin via the KERS complex provides the first mechanistic, chromatin-based understanding of how development is connected with small molecule synthesis in fungi