Functional analysis of the apple fruit microbiome based on shotgun metagenomic sequencing of conventional and organic orchard samples.

Fruits harbour abundant and diverse microbial communities that protect them from post-harvest pathogens. Identification of functional traits associated with a given microbiota can provide a better understanding of their potential influence. Here, we focused on the epiphytic microbiome of apple fruit. We suggest that shotgun metagenomic data can indicate specific functions carried out by different groups and provide information on their potential impact. Samples were collected from the surface of 'Golden Delicious' apples from four orchards that differ in their geographic location and management practice. Approximately 1 million metagenes were predicted based on a high-quality assembly. Functional profiling of the microbiome of fruits from orchards differing in their management practice revealed a functional shift in the microbiota. The organic orchard microbiome was enriched in pathways involved in plant defence activities; the conventional orchard microbiome was enriched in pathways related to the synthesis of antibiotics. The functional significance of the variations was explored using microbial network modelling algorithms to reveal the metabolic role of specific phylogenetic groups. The analysis identified several associations supported by other published studies. For example, the analysis revealed the nutritional dependencies of the Capnodiales group, including the Alternaria pathogen, on aromatic compounds.

Recent advances in research on biocontrol of postharvest fungal decay in apples.

Apple is the largest fruit crop produced in temperate regions and is a popular fruit worldwide. It is, however, susceptible to a variety of postharvest fungal pathogens, including Penicillium expansum, Botrytis cinerea, Botryosphaeria dothidea, Monilia spp., and Alternaria spp. Decays resulting from fungal infections severely reduce apple quality and marketable yield. Biological control utilizing bacterial and fungal antagonists is an eco-friendly and effective method of managing postharvest decay in horticultural crops. In the current review, research on the pathogenesis of major decay fungi and isolation of antagonists used to manage postharvest decay in apple is presented. The mode of action of postharvest biocontrol agents (BCAs), including recent molecular and genomic studies, is also discussed. Recent research on the apple microbiome and its relationship to disease management is highlighted, and the use of additives and physical treatments to enhance biocontrol efficacy of BCAs is reviewed. Biological control is a critical component of an integrated management system for the sustainable approaches to apple production. Additional research will be required to explore the feasibility of developing beneficial microbial consortia and novel antimicrobial compounds derived from BCAs for postharvest disease management, as well as genetic approaches, such as the use of CRISPR/Cas9 technology.

Infrared thermography of in situ natural freezing and mechanism of winter-thermonasty in Rhododendron maximum.

Evergreen leaves of Rhododendron species inhabiting temperate/montane climates are typically exposed to both high radiation and freezing temperatures during winter when photosynthetic biochemistry is severely inhibited. Cold-induced "thermonasty," that is, lamina rolling and petiole curling, can reduce the amount of leaf area exposed to solar radiation and has been associated with photoprotection in overwintering rhododendrons. The present study was conducted on natural, mature plantings of a cold-hardy and large-leaved thermonastic North American species (Rhododendron maximum) during winter freezes. Infrared thermography was used to determine initial sites of ice formation, patterns of ice propagation, and dynamics of the freezing process in leaves to understand the temporal and mechanistic relationship between freezing and thermonasty. Results indicated that ice formation in whole plants is initiated in the stem, predominantly in the upper portions, and propagates in both directions from the original site. Ice formation in leaves initially occurred in the vascular tissue of the midrib and then propagated into other portions of the vascular system/venation. Ice was never observed to initiate or propagate into palisade, spongy mesophyll, or epidermal tissues. These observations, together with the leaf- and petiole-histology, and a simulation of the rolling effect of dehydrated leaves using a cellulose-based, paper-bilayer system, suggest that thermonasty occurs due to anisotropic contraction of cell wall cellulose fibers of adaxial versus abaxial surface as the cells lose water to ice present in vascular tissues.

Evidence for host-microbiome co-evolution in apple.

Plants evolved in association with a diverse community of microorganisms. The effect of plant phylogeny and domestication on host-microbiome co-evolutionary dynamics are poorly understood. Here we examined the effect of domestication and plant lineage on the composition of the endophytic microbiome of 11 Malus species, representing three major groups: domesticated apple (M. domestica), wild apple progenitors, and wild Malus species. The endophytic community of M. domestica and its wild progenitors showed higher microbial diversity and abundance than wild Malus species. Heirloom and modern cultivars harbored a distinct community composition, though the difference was not significant. A community-wide Bayesian model revealed that the endophytic microbiome of domesticated apple is an admixture of its wild progenitors, with clear evidence for microbiome introgression, especially for the bacterial community. We observed a significant correlation between the evolutionary distance of Malus species and their microbiome. This study supports co-evolution between Malus species and their microbiome during domestication. This finding has major implications for future breeding programs and our understanding of the evolution of plants and their microbiomes.

Eco-friendly management of postharvest fungal decays in kiwifruit.

Kiwifruit is purchased by consumers worldwide and is increasing in demand. Unfortunately, kiwifruit is susceptible to postharvest decay caused by a variety of fungal pathogens, including Botrytis cinerea, Penicillium expansum, Alternaria alternata, Botryosphaeria dothidea, and Diaporthe spp. Among these pathogens, B. cinerea is the most prevalent and devastating. Infections by these fungal pathogens result in a deterioration in fruit quality and a reduction in marketable yield. Eco-friendly methods to control kiwifruit postharvest decay have been explored as alternatives to the use of synthetic fungicides. In this review, we provide an overview and discuss the virulence and pathogenesis of fungi that are causal agents of kiwifruit decay, especially B. cinerea, including recent molecular and genomic studies. Advances in pre- and postharvest measures for postharvest decay management, including biological control, physical applications, the use of natural compounds and plant hormones, and the use of combined methods, are also reviewed. Eco-friendly control measures are a critical component of an integrated management approach for sustainable production of kiwifruit. The need for further research on the use of microbial consortia for the management of postharvest diseases of kiwifruit is also discussed.

Pre- and postharvest measures used to control decay and mycotoxigenic fungi in potato (Solanum tuberosum L.) during storage.

Potato (Solanum tuberosum L.), a worldwide, staple food crop, is susceptible to postharvest rots caused by a variety of fungal pathogens, including Fusarium spp., Alternaria spp., Phytophthora infestans, Helminthosporium solani, Rhizoctonia solani, and Colletotrichum coccodes. Rots resulting from infections by these pathogens cause a significant reduction in potato quality and marketable yield. Importantly, some of these decay fungi also produce mycotoxins that represent a potential risk to human health. In the present review, an overview and discussion are provided on the epidemiology and pathogenesis of decay fungi, especially Fusarium spp., that include recent data derived from genomic and phylogenetic analyses. The biosynthesis and functional role of fungitoxic metabolites such as trichothecene mycotoxins and fusaric acid, produced in rotted potatoes are also reviewed. Advances in pre- and postharvest measures for rot management, especially eco-friendly methods including physical control, biological control, the use of natural compounds, and other agricultural management practices are also reviewed. Lastly, novel approaches to control potato dry rot such as the use of mycoviruses and CRISPR technology are highlighted.

Current and future trends in the biocontrol of postharvest diseases.

Postharvest diseases of fruits and vegetables cause significant economic losses to producers and marketing firms. Many of these diseases are caused by necrotrophic fungal pathogens that require wounded or injured tissues to establish an infection. Biocontrol of postharvest diseases is an evolving science that has moved from the traditional paradigm of one organism controlling another organism to viewing biocontrol as a system involving the biocontrol agent, the pathogen, the host, the physical environment, and most recently the resident microflora. Thus, the paradigm has shifted from one of simplicity to complexity. The present review provides an overview of how the field of postharvest biocontrol has evolved over the past 40 years, a brief review of the biology of necrotrophic pathogens, the discovery of BCAs, their commercialization, and mechanisms of action. Most importantly, current research on the use of marker-assisted-selection, the fruit microbiome and its relationship to the pathobiome, and the use of double-stranded RNA as a biocontrol strategy is discussed. These latter subjects represent evolving trends in postharvest biocontrol research and suggestions for future research are presented.

Experimental evidence of microbial inheritance in plants and transmission routes from seed to phyllosphere and root.

While the environment is considered the primary origin of the plant microbiome, the potential role of seeds as a source of transmitting microorganisms has not received much attention. Here we tested the hypothesis that the plant microbiome is partially inherited through vertical transmission. An experimental culturing device was constructed to grow oak seedlings in a microbe-free environment while keeping belowground and aboveground tissues separated. The microbial communities associated with the acorn's embryo and pericarp and the developing seeding's phyllosphere and root systems were analysed using amplicon sequencing of fungal ITS and bacterial 16S rDNA. Results showed that the seed microbiome is diverse and non-randomly distributed within an acorn. The microbial composition of the phyllosphere was diverse and strongly resembled the composition found in the embryo, whereas the roots and pericarp each had a less diverse and distinct microbial community. Our findings demonstrate a high level of microbial diversity and spatial partitioning of the fungal and bacterial community within both seed and seedling, indicating inheritance, niche differentiation and divergent transmission routes for the establishment of root and phyllosphere communities.

The impact of the endophytic bacterial community on mulberry tree growth in the Three Gorges Reservoir ecosystem, China.

Plant-associated microbes influence plant performance and may also impact biotic and abiotic stress tolerance. The microbiome of mulberry trees planted for ecological restoration in the hydro-fluctuation belt of the Three Gorges Reservoir Region, China, exhibited distinct patterns of localization. The endosphere exhibited lower α-diversity relative to the rhizosphere, but was more closely related to host growth status, especially in stem tissues. Pantoea was the predominant bacterial genus inhabiting the stems of two well-growing plants, while sequences identified as Pseudomonas and Pantoea were abundant in poorly growing plants. The complexity of the endophytic community was more connected to growth status in well-growing plants than it was in poorly growing plants. Among 151 endophytes cultured from collected samples of mulberry, 64 exhibited plant growth-promoting (PGP) potential in vitro and the majority of beneficial taxa were harvested from well-growing plants. Collectively, the present study indicates that the recruitment of beneficial endophytes may contribute to mulberry fitness under abiotic stress, and it provides a foundation for the development of a new strategy in vegetation restoration.

Global analysis of the apple fruit microbiome: are all apples the same?

We present the first worldwide study on the apple (Malus × domestica) fruit microbiome that examines questions regarding the composition and the assembly of microbial communities on and in apple fruit. Results revealed that the composition and structure of the fungal and bacterial communities associated with apple fruit vary and are highly dependent on geographical location. The study also confirmed that the spatial variation in the fungal and bacterial composition of different fruit tissues exists at a global level. Fungal diversity varied significantly in fruit harvested in different geographical locations and suggests a potential link between location and the type and rate of postharvest diseases that develop in each country. The global core microbiome of apple fruit was represented by several beneficial microbial taxa and accounted for a large fraction of the fruit microbial community. The study provides foundational information about the apple fruit microbiome that can be utilized for the development of novel approaches for the management of fruit quality and safety, as well as for reducing losses due to the establishment and proliferation of postharvest pathogens. It also lays the groundwork for studying the complex microbial interactions that occur on apple fruit surfaces.

The relationship of cold acclimation and extracellular ice formation to winter thermonasty in two Rhododendron species and their F1 hybrid.

PREMISE: Thermonastic leaf movements in evergreen Rhododendron species have been used to study plant strategies for winter photoprotection. To add to the current fundamental understanding of this behavior, we addressed the following questions: (1) Is the cold-acclimated (CA) state necessary for thermonasty, and do cold-induced leaf movements also occur in non-acclimated (NA) plants? (2) Which of the two movements, leaf rolling versus curling, is more responsive to freezing, if any, in a non-thermonastic species? (3) What is the temporal relationship between extracellular freezing and thermonasty? (4) What genetic inferences can be drawn from leaf movement in an F1 hybrid relative to its parents? METHODS: A temperature-controlled, gradual cooling regime was used to quantify freeze-induced leaf movements. Infrared thermography was used to confirm extracellular ice-formation in leaves. RESULTS: Both NA and CA plants of thermonastic species exhibited thermonasty, but leaf rolling/curling increased significantly in CA plants. In the cold-acclimated condition, a non-thermonastic species showed almost no rolling during freezing, while the thermonastic species and F1 hybrid did, the latter exhibiting a response intermediate to the parents. Freezing-induced leaf curling in the non-thermonastic species and the F1 hybrid was equivalent and significantly less than the degree of curling in the thermonastic species. CONCLUSIONS: Milder thermonasty in NA than CA leaves could be associated with differential anisotropy in the rolling forces and/or response of aquaporins to freezing. Leaf movements in the hybrid suggest that leaf rolling and curling are additive and dominant genetic traits, respectively. Infrared thermography confirms that ice formation in tissues precedes cold-induced thermonasty in R. catawbiense.

Fungal diversity in the coastal waters of King George Island (maritime Antarctica).

Fungi have been reported as common inhabitants of the maritime waters in Antarctica by studies based on culture-dependent methods. More recently, results obtained using DNA sequencing technologies, revealed that fungal diversity worldwide has been underestimated by culture methods. The present study provides the first characterization of fungal communities in the coastal waters of King George Island (maritime Antarctica) using both culture-dependent and high-throughput sequencing (HTS) methods. HTS demostrated a higher level of fungal diversity than the obtained by culture methods. A high prevalence of basidiomycetous yeasts and ascomycetous filamentous fungi was confirmed by both methods, however, Chythriomycota, Rozellomycota, lichenized fungi and Malassezia spp. were detected only by HTS. Correspondingly, members of some genera, such as Metschnikowia, were only found by culture-dependent methods. Our results confirm that culturing and HTS, should be seen as complementary approaches that enable one to obtain a more comprehensive picture of the composition of microbial communities.

Multiple transcriptomic analyses and characterization of pathogen-related core effectors and LysM family members reveal their differential roles in fungal growth and pathogenicity in Penicillium expansum.

Penicillium expansum is a destructive phytopathogen causing postharvest decay on many stored fruits. To develop effective and safe management strategies, it is important to investigate its pathogenicity-related mechanisms. In this study, a bioinformatic pipeline was constructed and 50 core effector genes were identified in P. expansum using multiple RNA-seq data sets and their putative functions were implicated by comparatively homologous analyses using pathogen-host interaction database. To functionally characterize P. expansum LysM domain proteins during infection, null mutants for the 15 uncharacterized putative LysM effectors were constructed and the fungal growth rate on either PDA or Cazpek medium or lesion expansion rate on the infected apple fruits was evaluated. The results showed the growth rate of knockout mutants from PeLysM5, PeLysM12 and PeLysM15 was retarded on PDA medium. No significant difference in growth rate was observed between wild type and all mutants on solid Cazpek medium. Nevertheless, the hypha of wild type displayed deeper yellow on the back of Cazpek medium than those of knockout mutants. On the infecting apples fruits, the knockout mutants from PeLysM5, PeLysM7, PeLysM8, PeLysM9, PeLysM10, PeLysM11, PeLysM14, PeLysM15, PeLysM16, PeLysM18 and PeLysM19 showed enhanced fungal virulence, with faster decaying on infected fruits than those from wild type. By contrast, the knockout mutation at PeLysM12 locus led to reduced lesion expansion rate on the infected apple fruits. In addition, P. expansum-apple interaction RNA-seq experiment was performed using apple fruit tissues infected by the wild type and knockout mutant ΔPeLysM15, respectively. Transcriptome analyses indicated that deletion of PeLysM15 could activate expression of several core effector genes, such as PEX2_055830, PEX2_036960 and PEX2_108150, and a chitin-binding protein, PEX2_064520. These results suggest PeLysM15 may play pivotal roles in fungal growth and development and involve pathogen-host interaction by modulating other effector genes' expression. Our results could provide solid data reference and good candidates for further pathogen-related studies in P. expansum.

Fox Hunting in Wild Apples: Searching for Novel Genes in Malus Sieversii.

Malus sieversii is considered the progenitor of modern apple (Malus pumila) cultivars and to represent a valuable source of genetic diversity. Despite the importance of M. sieversii as a source of disease resistance, stress tolerance, and novel fruit traits, little is known about gene function and diversity in M. sieversii. Notably, a publicly annotated genome sequence for this species is not available. In the current study, the FOX (Full-length cDNA OvereXpressing) gene hunting system was used to construct a library of transgenic lines of Arabidopsis in which each transgenic line overexpresses a full-length gene obtained from a cDNA library of the PI619283 accession of M. sieversii. The cDNA library was constructed from mRNA obtained from bark tissues collected in late fall-early winter, a time at which many abiotic stress-adaptative genes are expressed. Over 4000 apple FOX Arabidopsis lines have been established from the pool of transgenic seeds and cDNA inserts corresponding to various Gene Ontology (GO) categories have been identified. A total of 160 inserts appear to be novel, with no or limited homology to M. pumila, Arabidopsis, or poplar. Over 1300 lines have also been screened for freezing resistance. The constructed library of transgenic lines provides a valuable genetic resource for exploring gene function and diversity in Malus sieversii. Notably, no such library of t-DNA lines currently exists for any Malus species.

The impact of the postharvest environment on the viability and virulence of decay fungi.

Postharvest decay of fruits, vegetables, and grains by fungal pathogens causes significant economic losses. Infected produce presents a potential health risk since some decay fungi produce mycotoxins that are hazardous to human health. Infections are the result of the interplay between host resistance and pathogen virulence. Both of these processes, however, are significantly impacted by environmental factors, such as temperature, UV, oxidative stress, and water activity. In the present review, the impact of various physical postharvest treatments (e.g., heat and UV) on the viability and virulence of postharvest pathogens is reviewed and discussed. Oxidative injury, protein impairment, and cell wall degradation have all been proposed as the mechanisms by which these abiotic stresses reduce fungal viability and pathogenicity. The response of decay fungi to pH and the ability of pathogens to modulate the pH of the host environment also affect pathogenicity. The effects of the manipulation of the postharvest environment by ethylene, natural edible coatings, and controlled atmosphere storage on fungal viability are also discussed. Lastly, avenues of future research are proposed.

Biocontrol activity of a cold-adapted yeast from Tibet against gray mold in cherry tomato and its action mechanism.

Cold-adapted biocontrol yeast was selected from four yeast isolates from Tibet against gray mold of cherry tomato in cold storage. The strain numbered LB2 showed the best biocontrol activity and identified as Cryptococcus laurentii. Competition for nutrient, space, and induced fruit resistance was also its antagonistic mechanism. Compared with C. laurentii from sea-level place, the reason why LB2 had a better biocontrol activity was studied. More trehalose and proline in cell of LB2 made it exhibit a better cellular activity at low temperature, such as higher population dynamics in the wounds of cherry tomato and more biocontrol-related enzyme secretion, chitinase and ß-glucanase. The better oxidative stress tolerance was another characteristic of LB2. Maybe because of the ideal culture condition, there was no obvious difference between these two yeasts in the growth in vitro test at low temperature. Although the same phenomenon existed in the low pH stress test, LB2 still had higher cell concentration under this stress. Comparative transcriptomics method was also applied to analyze the cell activity of LB2 and C. laurentii at different temperatures. The results showed that more active response in the intracellular structure and intracellular metabolic process to cold temperature made LB2 had a better activity. The present study indicated a possibility to select cold-adapted biocontrol yeast from Tibet and also showed its primary action mechanism.

Potential Role of Exoglucanase Genes (WaEXG1 and WaEXG2) in the Biocontrol Activity of Wickerhamomyces anomalus.

The use of yeasts, including Wickerhamomyces anomalus, as biocontrol agents of fungi responsible for postharvest diseases of fruits and vegetables has been investigated for the past two decades. Among a variety of mechanisms, the production of glucanases coded by the "killer genes" WaEXG1 and WaEXG2 have been reported to play a role in the ability of yeast to inhibit other fungi. The objective of the present study was to determine the expression of these genes by RT-qPCR, utilizing gene-specific primers, when W. anomalus was grown on grape berries and oranges that were either non-inoculated or inoculated with Botrytis cinerea or Penicillium digitatum, or in minimal media supplemented with cell walls of various plant pathogens and different amounts of glucose. Results indicated that WaEXG2 was more responsive than WaEXG1 to the nutritional environment (including the addition of glucose to cell wall-amended media) in vitro and appeared to play a greater role in the cellular metabolism of W. anomalus. WaEXG2 expression also appeared to be more responsive to the presence of cell walls of P. digitatum and B. cinerea than other fungal species, whereas the same level of induction was not seen in vivo when the yeast was grown in wounded/pathogen-inoculated fruits.

Fire Blight Resistance in Wild Accessions of Malus sieversii.

Fire blight (Erwinia amylovora) is a devastating bacterial disease in apple that results in severe economic losses. Epidemics are becoming more common as susceptible cultivars and rootstocks are being planted, and control is becoming more difficult as antibiotic-resistant strains develop. Resistant germplasm currently being utilized by breeding programs tend to have small fruit size and poor flavor characteristics. Malus sieversii, a progenitor species of domestic apple, is notable for its relatively large, palatable fruit and some accessions have been reported to be resistant to fire blight. In this study, nearly 200 accessions of M. sieversii and appropriate controls were inoculated with E. amylovora in both Washington and West Virginia to identify fire blight resistant accessions. Twelve accessions were identified with resistance comparable to highly resistant and resistant controls. Several accessions exhibited a unique resistance response, not previously reported in domestic apple (M. × domestica), characterized by low incidence of infection but high severity once infection was initiated. Several of these M. sieversii accessions will be used as parents in future crosses in the Washington State University apple breeding program.

Engineering carpel-specific cold stress tolerance: a case study in Arabidopsis.

Climate change predictions forecast an increase in early spring frosts that could result in severe damage to perennial crops. For example, the Easter freeze of April 2007 left several states in the United States reporting a complete loss of that year's peach crop. The most susceptible organ to early frost damage in fruit trees is the carpel, particularly during bloom opening. In this study, we explored the use of a carpel-specific promoter (ZPT2-10) from petunia (Petunia hybrida var. Mitchell) to drive expression of the peach dehydrin PpDhn1. In peach, this gene is exceptionally responsive to low temperature but has not been observed to be expressed in carpels. This study examined carpel-specific properties of a petunia promoter driving the expression of the GUS gene (uidA) in transgenic Arabidopsis flowers and developed a carpel-specific ion leakage test to assess freezing tolerance. A homozygous Arabidopsis line (line 1-20) carrying the petunia ZPT2-10 promoter::PpDhn1 construct was obtained and freezing tolerance in the transgenic line was compared with an untransformed control. Overexpression of PpDhn1 in line 1-20 provided as much as a 1.9°C increase in carpel freezing tolerance as measured by electrolyte leakage.

Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity.

The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples. Li et al. ( MPMI-12-14-0398-FI ) reported similar results and conclusions in their recently accepted paper.