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.

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.

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.

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.

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.