Off-field practices have the advantage that land owners do not need to change their typical crop management; however, they still entail implementation and opportunity costs. On the other hand, on-field practices generally necessitate changes in how farmers manage their crops, which may reduce implementation costs but potentially increase opportunity costs . Occasionally, the costs of implementing these pollinator-supporting practices are higher than the income derived from their implementation , resulting in a low likelihood of adoption. However, such practices can generate other benefits for society, such as the enhancement of biodiversity; mitigation of soil erosion; and improvements in pest control, nutrient cycling, and/or water use efficiency . Many countries have therefore developed government sponsored programs that compensate farmers for enhancing biodiversity and ecosystem services, which are essential for human well-being but have no market value. In other situations, pollinator-supporting practices are profitable to farmers independent of government payments . Some of these practices imply lower costs or fewer additional costs . In the US state of Michigan, large square plant pots plantings of native wildflowers gradually increased wild bee and syrphid abundance as well as blueberry yield in fields adjacent to the plantings, as compared with fields with a standard grassy perimeter .
While the cost of establishing the plantings resulted in negative profit in the first year, the gain from pollinator enhanced yield outpaced the costs of the establishment and maintenance by the fourth year, and growers made cumulative profits . The plantings were on land that could not be cropped with blueberry because of soil or topography limitations, so there was no opportunity cost of “lost” crop production. The perennial wildflower plantings, if properly managed, will likely provide this benefit for many years. Furthermore, such practices have the added benefit of supplying habitat for natural enemies and enhancing biological control of pests in fields adjacent to the plantings. Although economic valuations of pollination services exist , studies that consider both the costs and benefits of pollinator-supporting practices are rare . We highlight the importance of estimating the marginal profits of implementing such practices , because management usually only partially increases or decreases ecosystem services .The fungal family Botryosphaeriaceae was introduced in 1918 by Theissen and Sydow with Botryosphaeria as the type genus. Members of this group have been taxonomically characterized based on the production of large, ovoid to oblong, typically hyaline, aseptate ascospores, which may become brown and septate with age, within bitunicate asci within unilocular or multilocular botryose ascomata known as pseudothecia . The asexual states of Botryosphaeriaceae exhibit a wide range of conidial morphologies that are taxonomically informative . Crous et al. contributed to stabilize the taxonomy of the genera within the Botryosphaeriaceae by employing a natural unit classification scheme, which is also referred to as the “genusfor-genus concept” .
The distinct asexual morphs were linked to unique sexual morphs on a unit-by-unit basis, which was corroborated with phylogenetic analysis of 28S rDNA sequence data revealing 10 generic clades. The Botryosphaeriaceae is currently composed of 24 well-defined genera and more than 200 species that are cosmopolitan in distribution and exist primarily as saprobes, endophytes, or pathogens on a wide array of important perennial plant hosts , in both human altered and natural ecosystems . The ecology of Botryosphaeriaceous taxa is complex and not fully understood. For example, in spite of being a shoot blight and canker pathogen of pine, Diplodia sapinea has been isolated from the bark surface and internal woody tissues of woody twigs from asymptomatic Pinus , representing what some may consider an “endophytic phase,” in which neither the internal plant tissues from which it is isolated, nor other plant tissues/organs showed apparent symptoms, nor were there negative impacts to host growth at the time of isolation. A similar pattern in the ecology of other Botryosphaeriaceaespecies considered pathogenic, but later being isolated during an endophytic phase, has been documented . In some cases, abiotic stress has been shown to induce severe symptoms in different host plants infected with seemingly innocuous Botryosphaeriaceae . This relationship between abiotic stress and more severe symptoms or more rapid colonization has also been reported for pathogenic species, e.g., Neofusiococcum parvum causing Botryosphaeria dieback of grape and Botryosphaeria dothidea causing Pistachio panicle and shoot blight .
Under climate-change scenarios of more frequent temperature extremes and prolonged drought, the interactions between host plants and Botryosphaeriaceae species may transit more readily from endophytic to pathogenic . An increase in Botryosphaeriaceae symptom severity in conjunction with other biotic stresses has also been documented in the literature . Members of the Botryosphaeriaceae are probably most well known as being destructive blight and canker pathogens of planted hosts . In agricultural settings, for example, they infect a large number of fruit and nut crops, such as almond , apple , avocado , citrus , grapevine , olive , pistachio , and walnut . In forest plantations in Australia and South Africa, for example, they infect Eucalyptus spp. and Pinus spp. . Infection is through either wounds to green and woody tissues or through natural openings in flowers, fruit, leaves, and shoots. The pathogens produce enzymes and/or toxins that kill cells and tissues of the various plant organs they attack. Infections of woody tissues of perennial hosts, either deep in the wood or just below the bark, can lead to stunted shoot growth, with eventual shoot death or “dieback.” Ecological genomic comparisons of phytopathogenic and saprobic fungi suggest that the former possess expanded gene families that generally fall into two main functional categories: lytic capabilities and putative transporters . Fungal lignin peroxidases, peroxidases, laccases, and polyphenol oxidases allow fungi to gain access to nutrients and to protect themselves from host defenses while growing in wood . Pathogenic species with the ability to enzymatically decompose a broader diversity of cell wall carbohydrates might be expected to more rapidly colonize, kill, and/or decompose host tissue. Membrane transporters of fungal plant pathogens also play important roles in exporting virulence factors involved in pathogenesis, influx of nutrients, and efflux of host-derived defense antimicrobial compounds . Previous genomic comparisons of phylogenetically diverse wood-infecting pathogens of grape revealed expansions in the repertoire of cell-wall degrading enzymes called carbohydrate-active enzyme gene families, whose protein products are involved in the synthesis, degradation, and/or modification of glycosidic bonds of plant cell wall constituents, including the main components of wood, cellulose, hemicelluloses, lignin , and significantly so in Neof. Further, a recent genomic annotation and in planta transcriptomic study of putative virulence factors of Neof. parvum during wood colonization revealed 567 protein-coding genes belonging to 52 different CAZyme families with glycoside hydrolases , which made up approximately 50% of the pathogen’s cell-wall degrading repertoire . Likewise, Yan et al. identified 820 CAZymes with at least 10 families that have experienced expansion in the genome of Lasiodiplodia theobromae with GHs representing the largest super family involved in the modification of plant cell wall carbohydrates. Genome comparisons of B. dothidea, L. theobromae, and Neof. parvum revealed that the genome of L. theobromae, the most virulent of the three species, is expanded in gene families associated with membrane transport, mainly ATP-binding cassette , large square planting pots and major facilitator super families. That same study reported 17 membrane transport genes that were significantly up-regulated upon host recognition including amino acid transporters and sugar porters. The largest transporter families reported in the genome of Neof. parvum include MFS, Peroxisomal Protein Importer family, and the ABC superfamily . In this study we analyze the genome sequences of seventeen Botryosphaeriaceae species representing six genera , which are wood-canker pathogens that attack horticultural crops, namely grape, pistachio, Prunus species , and walnut. Our objective is to examine through phylogenomic comparisons this comprehensive set of species on one host, grape, to better understand the evolutionary trends within this important fungal family, especially as it pertains to the gene space involving pathogenesis of woody tissues and fungal virulence.
All fungal isolates utilized in this study were obtained from internal wood cankers of symptomatic hosts following the protocol of Baumgartner et al. . Total genomic DNA was extracted following Morales-Cruz et al. . The internal transcribed spacer and translation elongation factor loci were amplified for each isolate via PCR using primers ITS5/ITS4 and EF1-688F/EF1-1251R . TEF and ITS sequences of each species were concatenated and aligned using MUSCLE v3.8.31 with default parameters. The alignment was cleaned with GBlocks v. 0.91b with a minimum block’s length of 5 bp and half of the gaps allowed. PhyML was used to calculate the maximum likelihood tree using 100 bootstrap replications, HKY85 substitution model and the subtree-pruning-regrafting method for searching for optimal tree topology. The resulting tree was visualized and edited for presentation using FigTree v1.4.1 .DNA extraction was done following the methods used by Morales-Cruz et al. using the axenic cultures of the isolated fungi and a CTAB protocol. Sequencing libraries were prepared and sequenced as described in Morales-Cruz et al. . After adapter ligation, libraries were size selected to 550–600 bp using a double-sided size selection with Ampure XP magnetic beads to remove unused adapter and adapter dimer. Sequencing was carried out on an Illumina HiSeq4000 machine at the DNA Technologies Core at UC Davis. Paired-end reads of 150 bp in length were generated. Raw reads were trimmed for quality and adapter removal using Trimmomatic v0.36 with options LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:100. Assembly of high quality reads was made using SPAdes v3.9 with the careful option and automatic read coverage cutoff. Assembly completeness was assessed using the Core Eukaryotic Genes Mapping Approach and Benchmarking Universal Single-Copy Orthologs analysis. Repeat Masker v.4.06 with default parameters was used to mask repeats. Gene model prediction was performed with Augustus v.3.2.1 with default parameters and using Neof. parvum gene model as training set. Sequencing data are available at NCBI . Sequencing data of Diplodia seriata and Neof. parvum can be retrieved from NCBI under BioProject PRJNA261773 and PRJNA321421, respectively. All genome assemblies and gene models are publicly available at Zenodo .The general annotation of the predicted proteins was assigned based on the similarities with peptides in the GenBank with Blast2GO , and to conserved domains in Pfam database . The functional annotation was assigned based on the databases and parameters presented in Supplementary Table 4. CAZymes were annotated with the dbCAN2 . The signal peptides were predicted using SignalP 5.0 . Secondary metabolites clusters were annotated using antiSMASH 5.0 . Peroxidases were annotated using a specialized database for fungi called fPoxDB . CYPED 6.0 was used to annotate the Cytochrome P450 proteins . At last, the proteins involved in transportation functions were annotated using the TCDB .Seventy-three single copy peptides used in Floudas et al. for fungal phylogeny reconstruction were extracted from the reference strain Saccharomyces cerevisiae 2S88C Genome Release 64-2-1 . All these peptides were compared using BLASTP against the seventeen Botryosphaeriaceae species and two wood-decay basidiomycetes that colonize grape: pathogenic, wood-rotting fungus Fomitiporia mediterranea and saprobic, white-rot fungus Stereum hirsutum. Fomitiporia mediterranea is one of a complex of pathogens that causes the grapevine trunk disease Esca in Europe, whereas the pathogenicity of St. hirsutum to grape is not known . Twenty-one proteins had exactly one top hit in all the species. The rest of the seventy-three initial proteins were excluded because they were either not present in all the species or had paralogs. Each set of orthologous proteins was aligned using MUSCLE v3.8.31 . Alignments were concatenated and cleaned using GBlocks v.0.91b , reducing the initial 21,008 positions to 12,066 informative positions. Clean alignments were imported into BEAUti v1.10.4 to prepare them for BEAST v1.10.4 analysis . Monophyletic partitions were set for Ascomycota, Basidiomycota, and Dothideomycetes species. Calibrations points were set to 588 and 350 mya on Ascomycota and Dothideomycetes partition, respectively, according to Beimforde et al. . Six MCMC chains of 1,000,000 steps were launched on BEAST . The resulting trees were concatenated with LogCombiner v1.10.4 and a consensus tree was obtained from TreeAnnotator v1.10.4 . FigTree v1.4.1 and Inkscape v.1.0.1 were used to edit the tree for figure presentation.The pathogenicity of 17 species of Botryosphaeriaceae was evaluated on potted grapevines in replicate experiments in the greenhouse . Hardwood cuttings obtained from a commercial nursery were propagated in May/June 2016 following the protocol of Travadon et al. . Inoculations took place after callusing and before planting in pots in the greenhouses. For inoculations, the commonly used agar plug method was employed .