Plant Seeds Commonly Host Bacillus spp., Potential Antagonists of Phytopathogens.

In agriculture, horticulture and plantation forestry, Bacillus species are the most commonly applied antagonists and biopesticides, targeting plant pathogens and insect pests, respectively. Bacillus isolates are also used as bacterial plant biostimulants, or BPBs. Such useful isolates of Bacillus are typically sourced from soil. Here, we show that Bacillus - and other antagonistic microbes - can be sourced from a broad range of plant seeds. We found that culturable Bacillus isolates are common in the seeds of 98 plant species representing 39 families (i.e., 87% of the commonly cultured bacteria belonged to Bacillales). We also found that 83% of the commonly cultured fungi from the seeds of the 98 plant species belonged to just three orders of fungi-Pleosporales, Hypocreales and Eurotiales-that are also associated with antagonism. Furthermore, we confirmed antagonism potential in agaro with seed isolates of Bacillus from Pinus monticola as a representative case. Eight isolates each of seed Bacillus, seed fungi, and foliar fungi, all from P. monticola, were paired in a total of 384 possible pair-wise interactions (with seed and foliar fungi as the targets). Seed Bacillus spp. were the strongest antagonists of the seed and foliar fungi, with a mean interaction strength 2.8 times greater than seed fungi (all either Eurotiales or Hypocreales) and 3.2 times greater than needle fungi. Overall, our study demonstrates that seeds host a taxonomically narrow group of culturable, antagonistic bacteria and fungi.

Facilitating Reforestation Through the Plant Microbiome: Perspectives from the Phyllosphere.

Tree planting and natural regeneration contribute to the ongoing effort to restore Earth's forests. Our review addresses how the plant microbiome can enhance the survival of planted and naturally regenerating seedlings and serve in long-term forest carbon capture and the conservation of biodiversity. We focus on fungal leaf endophytes, ubiquitous defensive symbionts that protect against pathogens. We first show that fungal and oomycetous pathogen richness varies greatly for tree species native to the United States (n = 0-876 known pathogens per US tree species), with nearly half of tree species either without pathogens in these major groups or with unknown pathogens. Endophytes are insurance against the poorly known and changing threat of tree pathogens. Next, we review studies of plant phyllosphere feedback, but knowledge gaps prevent us from evaluating whether adding conspecific leaf litter to planted seedlings promotes defensive symbiosis, analogous to adding soil to promote positive feedback. Finally, we discuss research priorities for integrating the plant microbiome into efforts to expand Earth's forests.

Culturable Seed Microbiota of Populus trichocarpa.

Plants harbor a diverse community of microbes, whose interactions with their host and each other can influence plant health and fitness. While microbiota in plant vegetative tissues has been extensively studied, less is known about members of the seed microbiota. We used culture-based surveys to identify bacteria and fungi found in the seeds of the model tree, Populus trichocarpa, collected from different sites. We found that individual P. trichocarpa seeds typically contained zero or one microbe, with common taxa including species of Cladosporium, Aureobasidium, Diaporthe, Alternaria, and Pseudomonas, a bacterium. Pseudomonas isolates were associated with seed mortality and were negatively associated with the occurrence of fungal isolates within Epicoccum, Alternaria, and Aureobasidium from the same seed. Next, we conducted an inoculation experiment with one of the isolated seed microbes, Pseudomonas syringae pv. syringae, and found that it reduced seed germination and increased seedling mortality for P. trichocarpa. Our findings highlight common fungi and bacteria in the seeds of P. trichocarpa, prompting further study of their functional consequences. Moreover, our study confirms that P. syringae pv. syringae is a seed pathogen of P. trichocarpa and is the first report that P. syringae pv. syringae is a lethal seedling pathogen of P. trichocarpa, allowing for future work on the pathogenicity of this bacterium in seedlings and potential antagonism with other seed microbes.

Leaf Endophytes of Populus trichocarpa Act as Pathogens of Neighboring Plant Species.

The conventional definition of endophytes is that they do not cause disease, whereas pathogens do. Complicating this convention, however, is the poorly explored phenomenon that some microbes are endophytes in some plants but pathogens in others. Black cottonwood or poplar (Populus trichocarpa) and wheat (Triticum aestivum) are common wild and crop plants, respectively, in the Pacific Northwest USA. The former anchors wild, riparian communities, whereas the latter is an introduced domesticate of commercial importance in the region. We isolated Fusarium culmorum - a well-known pathogen of wheat causing both blight and rot - from the leaf of a black cottonwood tree in western Washington. The pathogenicity of this cottonwood isolate and of a wheat isolate of F. culmorum were compared by inoculating both cottonwood and wheat in a greenhouse experiment. We found that both the cottonwood and wheat isolates of F. culmorum significantly reduced the growth of wheat, whereas they had no impact on cottonwood growth. Our results demonstrate that the cottonwood isolate of F. culmorum is endophytic in one plant species but pathogenic in another. Using sequence-based methods, we found an additional 56 taxa in the foliar microbiome of cottonwood that matched the sequences of pathogens of other plants of the region. These sequence-based findings suggest, though they do not prove, that P. trichocarpa may host many additional pathogens of other plants.

Isolation, Characterization, and Pathogenicity of Two Pseudomonas syringae Pathovars from Populus trichocarpa Seeds.

Pseudomonas syringae is a ubiquitous plant pathogen, infecting both woody and herbaceous plants and resulting in devastating agricultural crop losses. Characterized by a remarkable specificity for plant hosts, P. syringae pathovars utilize a number of virulence factors including the type III secretion system and effector proteins to elicit disease in a particular host species. Here, two Pseudomonas syringae strains were isolated from diseased Populustrichocarpa seeds. The pathovars were capable of inhibiting poplar seed germination and were selective for the Populus genus. Sequencing of the newly described organisms revealed similarity to phylogroup II pathogens and genomic regions associated with woody host-associated plant pathogens, as well as genes for specific virulence factors. The host response to infection, as revealed through metabolomics, is the induction of the stress response through the accumulation of higher-order salicylates. Combined with necrosis on leaf surfaces, the plant appears to quickly respond by isolating infected tissues and mounting an anti-inflammatory defense. This study improves our understanding of the initial host response to epiphytic pathogens in Populus and provides a new model system for studying the effects of a bacterial pathogen on a woody host plant in which both organisms are fully genetically sequenced.

Contingency rules for pathogen competition and antagonism in a genetically based, plant defense hierarchy.

Plant defense against pathogens includes a range of mechanisms, including, but not limited to, genetic resistance, pathogen-antagonizing endophytes, and pathogen competitors. The relative importance of each mechanism can be expressed in a hierarchical view of defense. Several recent studies have shown that pathogen antagonism is inconsistently expressed within the plant defense hierarchy. Our hypothesis is that the hierarchy is governed by contingency rules that determine when and where antagonists reduce plant disease severity.Here, we investigated whether pathogen competition influences pathogen antagonism using Populus as a model system. In three independent field experiments, we asked whether competition for leaf mesophyll cells between a Melampsora rust pathogen and a microscopic, eriophyid mite affects rust pathogen antagonism by fungal leaf endophytes. The rust pathogen has an annual, phenological disadvantage in competition with the mite because the rust pathogen must infect its secondary host in spring before infecting Populus. We varied mite-rust competition by utilizing Populus genotypes characterized by differential genetic resistance to the two organisms. We inoculated plants with endophytes and allowed mites and rust to infect plants naturally.Two contingency rules emerged from the three field experiments: (a) Pathogen antagonism by endophytes can be preempted by host genes for resistance that suppress pathogen development, and (b) pathogen antagonism by endophytes can secondarily be preempted by competitive exclusion of the rust by the mite. Synthesis: Our results point to a Populus defense hierarchy with resistance genes on top, followed by pathogen competition, and finally pathogen antagonism by endophytes. We expect these rules will help to explain the variation in pathogen antagonism that is currently attributed to context dependency.

Effects of Antagonists on Mycotoxins of Seedborne Fusarium spp. in Sweet Corn.

Fusarium species coexist as toxigenic, systemic pathogens in sweet corn seed production in southwestern Idaho, USA. We hypothesized that fungal antagonists of seedborne Fusarium would differentially alter production of Fusarium mycotoxins directly and/or systemically. We challenged the Fusarium complex by in vitro antagonism trials and in situ silk and seed inoculations with fungal antagonists. Fungal antagonists reduced growth and sporulation of Fusarium species in vitro from 40.5% to as much as 100%. Pichia membranifaciens and Penicillium griseolum reduced fumonisin production by F. verticillioides by 73% and 49%, respectively, while P. membranifaciens and a novel Penicillium sp. (WPT) reduced fumonisins by F. proliferatum 56% and 78%, respectively. In situ, pre-planting inoculation of seeds with Penicillium WPT systemically increased fumonisins in the resulting crop. Morchella snyderi applied to silks of an F1 cross systemically reduced deoxynivalenol by 47% in mature seeds of the F2. Antagonists failed to suppress Fusarium in mature kernels following silk inoculations, although the ratio of F. verticillioides to total Fusarium double with some inoculants. Fusarium mycotoxin concentrations in sweet corn seed change systemically, as well as locally, in response to the presence of fungal antagonists, although in Fusarium presence in situ was not changed.

Resistance to an eriophyid mite in an interspecific hybrid pedigree of Populus.

Schizoempodium mesophyllincola is an eriophyid mite that feeds in leaves of Populus trichocarpa in the central part of this cottonwood tree's range (i.e., coastal British Columbia, Washington and Oregon) in the Pacific Northwest (PNW) of North America, and on some interspecific hybrids planted in short-rotation, intensive forestry in the region. The mite, a leaf vagrant, sucks the contents of spongy mesophyll cells, causing leaf discoloration, or "bronzing." Here, we investigate the inheritance pattern of resistance to leaf bronzing using a three-generation Populus trichocarpa x P. deltoides hybrid pedigree. We found that resistance to the mite is an exaptation in that its source in two related F2 families of the TxD hybrid pedigree was the non-native host, P. deltoides. Two grandparental genotypes of the latter, 'ILL-5' and 'ILL-129', were completely free of the bronzing symptom and that phenotype was inherited in a Mendelian manner in the F1 and F2. Resistance to S. mesophyllincola is similar to resistance to many other regional pathogens of P. trichocarpa (e.g., Melampsora occidentalis, Venturia inopina, Sphaerulina populicola, and Taphrina sp.) in that it is inherited from the non-native grandparent (e.g., P. deltoides, P. nigra, or P. maximowiczii) in three-generation, hybrid pedigrees. In addition to finding evidence for Mendelian inheritance, we found two QTLs with LOD scores 5.03 and 3.12 mapped on linkage groups (LG) III and I, and they explained 6.7 and 4.2% of the phenotypic variance, respectively. The LG I QTL is close to, or synonymous with, one for resistance to sap-feeding arthropods and leaf developmental traits as expressed in a British study utilizing the same pedigree.

A Hypothetical Bottleneck in the Plant Microbiome.

The plant microbiome may be bottlenecked at the level of endophytes of individual seeds. Strong defense of developing seeds is predicted by optimal defense theory, and we have experimentally demonstrated exclusionary interactions among endophytic microbes infecting individual seeds of Centaurea stoebe. Having found a single, PDA-culturable microbe per seed or none in an exploratory study with Centaurea stoebe, we completed a more extensive survey of an additional 98 plant species representing 39 families. We again found that individual, surface-sterilized seeds of all species hosted only one PDA-culturable bacterial or fungal endophyte per seed, or none. PDA-unculturables were not determined but we expect them to also be bottlenecked in individual seeds, as they too should be governed by exclusionary interactions. If the bottleneck were confirmed with high-throughput sequencing of individual seeds then it would make sense to further investigate the Primary Symbiont Hypothesis (PSH). This includes the prediction that primary symbionts (i.e., the winners of the exclusionary battles among seed endophytes) have strong effects on seedlings depending on symbiont identity.

Sydowia polyspora is both a Foliar Endophyte and a Preemergent Seed Pathogen in Pinus ponderosa.

Poor seedling performance and reduced seed emergence are often ascribed to known pathogens that cause low seedling recruitment and poor seed emergence in forest nurseries and regeneration plantings. On the other hand, foliar endophytes are often overlooked as a source of poor emergence or tree seedling disease. Here, we show that an endophytic fungus common to the foliar microbiome of Pinus ponderosa acts as a cryptic pathogen in delaying emergence. In a series of experiments, we inoculated seed of P. ponderosa with a suspension of Sydowia polyspora 12 h prior to sowing. S. polyspora reduced seed emergence of its host, P. ponderosa, by as much as 30%. A tetrazolium chloride viability assay showed that S. polyspora reduces emergence by preventing germination; seed remained viable. In sum, pathogens affecting tree seed emergence and seedling recruitment may be endophytic as well as in seed and soil and deserve greater attention in studies of natural regeneration.

Morphological and Molecular Characterization of Two Aphelenchoides Endophytic in Poplar Leaves.

During a long-term, large network study of the ecology of plant endophytes in native habitats, various nematodes have been found. Two poplar species, Populus angustifolia (narrowleaf cottonwood) and Populus trichocarpa (black cottonwood), are important ecological and genomic models now used in ongoing plant-pathogen-endophyte interaction studies. In this study, two different aphelenchid nematodes within surface-sterilized healthy leaves of these two Populus spp. in northwestern North America were discovered. Nematodes were identified and characterized microscopically and molecularly with 28S ribosomal RNA (rRNA) and 18S rRNA molecular markers. From P. angustifolia, Aphelenchoides saprophilus was inferred to be closest to another population of A. saprophilus among sequenced taxa in the 18S tree. From P. trichocarpa, Laimaphelenchus heidelbergi had a 28S sequence only 1 bp different from that of a Portuguese population, and 1 bp different from the original Australian type population. The 28S and 18S rRNA trees of Aphelenchoides and Laimaphelenchus species indicated L. heidelbergi failed to cluster with three other Laimaphelenchus species, including the type species of the genus. Therefore, we support a conservative definition of the genus Laimaphelenchus, and consider these populations to belong to Aphelenchoides, amended as Aphelenchoides heidelbergi n. comb. This is the first report of these nematode species from within aboveground leaves. The presence of these fungal-feeding nematodes can affect the balance of endophytic fungi, which are important determinants of plant health.

Revisiting the Life Cycle of Dung Fungi, Including Sordaria fimicola.

Dung fungi, such as Sordaria fimicola, generally reproduce sexually with ascospores discharged from mammalian dung after passage through herbivores. Their life cycle is thought to be obligate to dung, and thus their ascospores in Quaternary sediments have been interpreted as evidence of past mammalian herbivore activity. Reports of dung fungi as endophytes would seem to challenge the view that they are obligate to dung. However, endophyte status is controversial because surface-sterilization protocols could fail to kill dung fungus ascospores stuck to the plant surface. Thus, we first tested the ability of representative isolates of three common genera of dung fungi to affect plant growth and fecundity given that significant effects on plant fitness could not result from ascospores merely stuck to the plant surface. Isolates of S. fimicola, Preussia sp., and Sporormiella sp. reduced growth and fecundity of two of three populations of Bromus tectorum, the host from which they had been isolated. In further work with S. fimicola we showed that inoculations of roots of B. tectorum led to some colonization of aboveground tissues. The same isolate of S. fimicola reproduced sexually on inoculated host plant tissues as well as in dung after passage through sheep, thus demonstrating a facultative rather than an obligate life cycle. Finally, plants inoculated with S. fimicola were not preferred by sheep; preference had been expected if the fungus were obligate to dung. Overall, these findings make us question the assumption that these fungi are obligate to dung.

Common foliar fungi of Populus trichocarpa modify Melampsora rust disease severity.

Nonpathogenic foliar fungi (i.e. endophytes and epiphytes) can modify plant disease severity in controlled experiments. However, experiments have not been combined with ecological studies in wild plant pathosystems to determine whether disease-modifying fungi are common enough to be ecologically important. We used culture-based methods and DNA sequencing to characterize the abundance and distribution of foliar fungi of Populus trichocarpa in wild populations across its native range (Pacific Northwest, USA). We conducted complementary, manipulative experiments to test how foliar fungi commonly isolated from those populations influence the severity of Melampsora leaf rust disease. Finally, we examined correlative relationships between the abundance of disease-modifying foliar fungi and disease severity in wild trees. A taxonomically and geographically diverse group of common foliar fungi significantly modified disease severity in experiments, either increasing or decreasing disease severity. Spatial patterns in the abundance of some of these foliar fungi were significantly correlated (in predicted directions) with disease severity in wild trees. Our study reveals that disease modification is an ecological function shared by common foliar fungal symbionts of P. trichocarpa. This finding raises new questions about plant disease ecology and plant biodiversity, and has applied potential for disease management.

Fungal endophytes: modifiers of plant disease.

Many recent studies have demonstrated that non-pathogenic fungi within plant microbiomes, i.e., endophytes ("endo" = within, "phyte" = plant), can significantly modify the expression of host plant disease. The rapid pace of advancement in endophyte ecology warrants a pause to synthesize our understanding of endophyte disease modification and to discuss future research directions. We reviewed recent literature on fungal endophyte disease modification, and here report on several emergent themes: (1) Fungal endophyte effects on plant disease span the full spectrum from pathogen antagonism to pathogen facilitation, with pathogen antagonism most commonly reported. (2) Agricultural plant pathosystems are the focus of research on endophyte disease modification. (3) A taxonomically diverse group of fungal endophytes can influence plant disease severity. And (4) Fungal endophyte effects on plant disease severity are context-dependent. Our review highlights the importance of fungal endophytes for plant disease across a broad range of plant pathosystems, yet simultaneously reveals that complexity within plant microbiomes presents a significant challenge to disentangling the biotic environmental factors affecting plant disease severity. Manipulative studies integrating eco-evolutionary approaches with emerging molecular tools will be poised to elucidate the functional importance of endophytes in natural plant pathosystems that are fundamental to biodiversity and conservation.

Genetics-based interactions among plants, pathogens, and herbivores define arthropod community structure.

Plant resistance to pathogens or insect herbivores is common, but its potential for indirectly influencing plant-associated communities is poorly known. Here, we test whether pathogens' indirect effects on arthropod communities and herbivory depend on plant resistance to pathogens and/or herbivores, and address the overarching interacting foundation species hypothesis that genetics-based interactions among a few highly interactive species can structure a much larger community. In a manipulative field experiment using replicated genotypes of two Populus species and their interspecific hybrids, we found that genetic variation in plant resistance to both pathogens and insect herbivores modulated the strength of pathogens' indirect effects on arthropod communities and insect herbivory. First, due in part to the pathogens' differential impacts on leaf biomass among the two Populus species and the hybrids, the pathogen most strongly impacted arthropod community composition, richness, and abundance on the pathogen-susceptible tree species. Second, we found similar patterns comparing pathogen-susceptible and pathogen-resistant genotypes within species. Third, within a plant species, pathogens caused a fivefold greater reduction in herbivory on insect-herbivore-susceptible plant genotypes than on herbivore-resistant genotypes, demonstrating that the pathogen-herbivore interaction is genotype dependent. We conclude that interactions among plants, pathogens, and herbivores can structure multitrophic communities, supporting the interacting foundation species hypothesis. Because these interactions are genetically based, evolutionary changes in genetic resistance could result in ecological changes in associated communities, which may in turn feed back to affect plant fitness.

Fungal endophyte increases the allelopathic effects of an invasive forb.

Endophytic plant symbionts can have powerful effects on the way their hosts interact with pathogens, competitors, and consumers. The presence of endophytes in plants can alter food webs, community composition and ecosystem processes, suggesting that endophyte-plant symbioses may represent unique forms of extended phenotypes. We tested the impact of the fungal endophyte Alternaria alternata (phylotype CID 120) on the allelopathic effect of the invasive forb Centaurea stoebe when in competition with the North American native bunchgrass Koeleria macrantha in a greenhouse competition experiment. The allelopathic effect of C. stoebe on K. macrantha when infected with the fungal endophyte was more than twice that of endophyte-free C. stoebe. However, this allelopathic effect was a small part of the very large competitive effect of C. stoebe on K. macrantha in all treatments, likely because of the priority effects in our experimental design. To our knowledge, these results are the first experimental evidence for a symbiotic relationship between plants and fungal endophytes affecting allelopathic interactions between competing plants, and thus provide insight into the mechanisms by which fungal endophytes may increase the competitive ability of their hosts.

Exclusionary interactions among diverse fungi infecting developing seeds of Centaurea stoebe.

Developing seeds are expected to be strongly defended against microbial attack. In keeping with this, only 26% of seeds of Centaurea stoebe from its native and invaded ranges in Eurasia and North America were infected with fungi, and 92.2% of those were infected with a single fungus per seed. Even when developing seeds in flower heads were inoculated under conducive conditions for infection with 14 of these seed-infecting fungi, re-isolation of inoculants was only 16% overall, and again limited to the particular inoculant. Environmental fungi (i.e. those not isolated from seed of C. stoebe) were present in control flower heads under conditions conducive to infection but they were never re-isolated from fully developed seeds in any experiments. When two or three seed isolates were co-inoculated to compete in flower heads, only one inoculant, and always the same one, was re-isolated from all matured seeds, regardless of maternal plant genotype. PCR-based detection methods confirmed that these fungal interactions were exclusionary rather than suppressive. In these strongly defended, developing seeds, we had expected the plant to control not only the overall level of infection but also the outcome of co-inoculations. Consequences for the next plant generation of this exclusionary competition among seed-infecting fungi included effects on seedling emergence, growth and fecundity.