Manish N. Raizada

Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology.

Endophytes are non-pathogenic microbes living inside plants. We asked whether endophytic species were conserved in the agriculturally important plant genus Zea as it became domesticated from its wild ancestors (teosinte) to modern maize (corn) and moved from Mexico to Canada. Kernels from populations of four different teosintes and 10 different maize varieties were screened for endophytic bacteria by culturing, cloning and DNA fingerprinting using terminal restriction fragment length polymorphism (TRFLP) of 16S rDNA. Principle component analysis of TRFLP data showed that seed endophyte community composition varied in relation to plant host phylogeny. However, there was a core microbiota of endophytes that was conserved in Zea seeds across boundaries of evolution, ethnography and ecology. The majority of seed endophytes in the wild ancestor persist today in domesticated maize, though ancient selection against the hard fruitcase surrounding seeds may have altered the abundance of endophytes. Four TRFLP signals including two predicted to represent Clostridium and Paenibacillus species were conserved across all Zea genotypes, while culturing showed that Enterobacter, Methylobacteria, Pantoea and Pseudomonas species were widespread, with γ-proteobacteria being the prevalent class. Twenty-six different genera were cultured, and these were evaluated for their ability to stimulate plant growth, grow on nitrogen-free media, solubilize phosphate, sequester iron, secrete RNAse, antagonize pathogens, catabolize the precursor of ethylene, produce auxin and acetoin/butanediol. Of these traits, phosphate solubilization and production of acetoin/butanediol were the most commonly observed. An isolate from the giant Mexican landrace Mixteco, with 100% identity to Burkholderia phytofirmans, significantly promoted shoot potato biomass. GFP tagging and maize stem injection confirmed that several seed endophytes could spread systemically through the plant. One seed isolate, Enterobacter asburiae, was able to exit the root and colonize the rhizosphere. Conservation and diversity in Zea-microbe relationships are discussed in the context of ecology, crop domestication, selection and migration.

The Diversity of Anti-Microbial Secondary Metabolites Produced by Fungal Endophytes: An Interdisciplinary Perspective

Endophytes are microbes that inhabit host plants without causing disease and are reported to be reservoirs of metabolites that combat microbes and other pathogens. Here we review diverse classes of secondary metabolites, focusing on anti-microbial compounds, synthesized by fungal endophytes including terpenoids, alkaloids, phenylpropanoids, aliphatic compounds, polyketides, and peptides from the interdisciplinary perspectives of biochemistry, genetics, fungal biology, host plant biology, human and plant pathology. Several trends were apparent. First, host plants are often investigated for endophytes when there is prior indigenous knowledge concerning human medicinal uses (e.g., Chinese herbs). However, within their native ecosystems, and where investigated, endophytes were shown to produce compounds that target pathogens of the host plant. In a few examples, both fungal endophytes and their hosts were reported to produce the same compounds. Terpenoids and polyketides are the most purified anti-microbial secondary metabolites from endophytes, while flavonoids and lignans are rare. Examples are provided where fungal genes encoding anti-microbial compounds are clustered on chromosomes. As different genera of fungi can produce the same metabolite, genetic clustering may facilitate sharing of anti-microbial secondary metabolites between fungi. We discuss gaps in the literature and how more interdisciplinary research may lead to new opportunities to develop bio-based commercial products to combat global crop and human pathogens.

Genetic diversity and genomic resources available for the small millet crops to accelerate a New Green Revolution

Small millets are nutrient-rich food sources traditionally grown and consumed by subsistence farmers in Asia and Africa. They include finger millet (Eleusine coracana), foxtail millet (Setaria italica), kodo millet (Paspalum scrobiculatum), proso millet (Panicum miliaceum), barnyard millet (Echinochloa spp.), and little millet (Panicum sumatrense). Local farmers value the small millets for their nutritional and health benefits, tolerance to extreme stress including drought, and ability to grow under low nutrient input conditions, ideal in an era of climate change and steadily depleting natural resources. Little scientific attention has been paid to these crops, hence they have been termed “orphan cereals.” Despite this challenge, an advantageous quality of the small millets is that they continue to be grown in remote regions of the world which has preserved their biodiversity, providing breeders with unique alleles for crop improvement. The purpose of this review, first, is to highlight the diverse traits of each small millet species that are valued by farmers and consumers which hold potential for selection, improvement or mechanistic study. For each species, the germplasm, genetic and genomic resources available will then be described as potential tools to exploit this biodiversity. The review will conclude with noting current trends and gaps in the literature and make recommendations on how to better preserve and utilize diversity within these species to accelerate a New Green Revolution for subsistence farmers in Asia and Africa.

Interactions between Co-Habitating fungi Elicit Synthesis of Taxol from an Endophytic Fungus in Host Taxus Plants

Within a plant, there can exist an ecosystem of pathogens and endophytes, the latter described as bacterial and fungal inhabitants that thrive without causing disease to the host. Interactions between microbial inhabitants represent a novel area of study for natural products research. Here we analyzed the interactions between the fungal endophytes of Taxus (yew) trees. Fungal endophytes of Taxus have been proposed to produce the terpenoid secondary metabolite, Taxol, an anti-cancer drug. It is widely reported that plant extracts stimulate endophytic fungal Taxol production, but the underlying mechanism is not understood. Here, Taxus bark extracts stimulated fungal Taxol production 30-fold compared to a 10-fold induction with wood extracts. However, candidate plant-derived defense compounds (i.e., salicylic acid, benzoic acid) were found to act only as modest elicitors of fungal Taxol production from the endophytic fungus Paraconiothyrium SSM001, consistent with previous studies. We hypothesized the Taxus plant extracts may contain elicitors derived from other microbes inhabiting these tissues. We investigated the effects of co-culturing SSM001 with other fungi observed to inhabit Taxus bark, but not wood. Surprisingly, co-culture of SSM001 with a bark fungus (Alternaria) caused a ∼threefold increase in Taxol production. When SSM001 was pyramided with both the Alternaria endophyte along with another fungus (Phomopsis) observed to inhabit Taxus, there was an ∼eightfold increase in fungal Taxol production from SSM001. These results suggest that resident fungi within a host plant interact with one another to stimulate Taxol biosynthesis, either directly or through their metabolites. More generally, our results suggest that endophyte secondary metabolism should be studied in the context of its native ecosystem.

Novel temporal, fine-scale and growth variation phenotypes in roots of adult-stage maize (Zea mays L.) in response to low nitrogen stress

There is interest in discovering root traits associated with acclimation to nutrient stress. Large root systems, such as in adult maize, have proven difficult to be phenotyped comprehensively and over time, causing target traits to be missed. These challenges were overcome here using aeroponics, a system where roots grow in the air misted with a nutrient solution. Applying an agriculturally relevant degree of low nitrogen (LN) stress, 30-day-old plants responded by increasing lengths of individual crown roots (CRs) by 63%, compensated by a 40% decline in CR number. LN increased the CR elongation rate rather than lengthening the duration of CR growth. Only younger CR were significantly responsive to LN stress, a novel finding. LN shifted the root system architectural balance, increasing the lateral root (LR)-to-CR ratio, adding ∼70 m to LR length. LN caused a dramatic increase in second-order LR density, not previously reported in adult maize. Despite the near-uniform aeroponics environment, LN induced increased variation in the relative lengths of opposing LR pairs. Large-scale analysis of root hairs (RHs) showed that LN decreased RH length and density. Time-course experiments suggested the RH responses may be indirect consequences of decreased biomass/demand under LN. These results identify novel root traits for genetic dissection.

Bacterial populations in juvenile maize rhizospheres originate from both seed and soil

Background and aimsTo assess the impacts of soil microbes and plant genotype on the composition of maize associated bacterial communities.MethodsTwo genotypes of Brazilian maize were planted indoors on sterile sand, a deep underground subsoil, and a nutrient-rich topsoil from the Amazon jungle (terra preta). DNA was extracted from rhizospheres, phyllospheres, and surface sterilized roots for 16S rDNA fingerprinting and next generation sequencing.ResultsNeither plant genotype nor soil type appeared to influence bacterial diversity in phyllospheres or endospheres. Rhizospheres showed strikingly similar 16S rDNA ordination of both fingerprinting and sequencing data, with soil type driving grouping patterns and genotype having a significant impact only on sterile sand. Rhizospheres grown in non-sterile soils contained greater bacterial diversity than sterile-sand grown ones, however the dominant OTUs (species of Proteobacteria and Bacteroidetes) were found in all rhizospheres suggesting seeds as a common source of inoculum. Rhizospheres of the commercial hybrid appeared to contain less bacterial diversity than the landrace.ConclusionsMaize rhizospheres receive diverse bacteria from soil, are influenced by the genotype or treatment of the seed, and are dominated by species of Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. As many dominant 16S rDNA sequences were observed in rhizospheres grown in both sterile and non-sterile substrate, we conclude that the most common bacterial cells in juvenile maize rhizospheres are seed transmitted.

Controlling weeds with fungi, bacteria and viruses: a review

Weeds are a nuisance in a variety of land uses. The increasing prevalence of both herbicide resistant weeds and bans on cosmetic pesticide use has created a strong impetus to develop novel strategies for controlling weeds. The application of bacteria, fungi and viruses to achieving this goal has received increasingly great attention over the last three decades. Proposed benefits to this strategy include reduced environmental impact, increased target specificity, reduced development costs compared to conventional herbicides and the identification of novel herbicidal mechanisms. This review focuses on examples from North America. Among fungi, the prominent genera to receive attention as bioherbicide candidates include Colletotrichum, Phoma, and Sclerotinia. Among bacteria, Xanthomonas and Pseudomonas share this distinction. The available reports on the application of viruses to controlling weeds are also reviewed. Focus is given to the phytotoxic mechanisms associated with bioherbicide candidates. Achieving consistent suppression of weeds in field conditions is a common challenge to this control strategy, as the efficacy of a bioherbicide candidate is generally more sensitive to environmental variation than a conventional herbicide. Common themes and lessons emerging from the available literature in regard to this challenge are presented. Additionally, future directions for this crop protection strategy are suggested.

Impact of swapping soils on the endophytic bacterial communities of pre-domesticated, ancient and modern maize

BackgroundEndophytes are microbes that live within plants such as maize (corn, Zea mays L.) without causing disease. It is generally assumed that most endophytes originate from soil. If this is true, then as humans collected, domesticated, bred and migrated maize globally from its native Mexico, they moved the species away from its native population of endophyte donors. The migration of maize persists today, as breeders collect wild and exotic seed (as sources of diverse alleles) from sites of high genetic diversity in Mexico for breeding programs on distant soils. When transported to new lands, it is unclear whether maize permits only selective colonization of microbes from the Mexican soils on which it co-evolved, tolerates shifts in soil-derived endophytes, or prevents colonization of soil-based microbes in favour of seed-transmitted microbes. To test these hypotheses, non-sterilized seeds of three types of maize (pre-domesticated-Mexican, ancient-Mexican, modern-temperate) were planted side-by-side on indigenous Mexican soil, Canadian temperate soil or sterilized sand. The impact of these soil swaps on founder bacterial endophyte communities was tested using 16S-rDNA profiling, culturing and microbial trait phenotyping.ResultsMultivariate analysis showed that bacterial 16S-rDNA TRFLP profiles from young, surface-sterilized maize plants were more similar when the same host genotype was grown on the different soils than when different maize genotypes were grown on the same soil. There appeared to be two reasons for this result. First, the largest fraction of bacterial 16S-signals from soil-grown plants was shared with parental seeds and/or plants grown on sterilized sand, suggesting significant inheritance of candidate endophytes. The in vitro activities of soil-derived candidate endophytes could be provided by bacteria that were isolated from sterile sand grown plants. Second, many non-inherited 16S-signals from sibling plants grown on geographically-distant soils were shared with one another, suggesting maize can select microbes with similar TRFLP peak sizes from diverse soils. Wild, pre-domesticated maize did not possess more unique 16S-signals when grown on its native Mexican soil than on Canadian soil, pointing against long-term co-evolutionary selection. The modern hybrid did not reject more soil-derived 16S-signals than did ancestral maize, pointing against such rejection as a mechanism that contributes to yield stability across environments. A minor fraction of 16S-signals was uniquely associated with any one soil.ConclusionWithin the limits of TRFLP profiling, the candidate bacterial endophyte populations of pre-domesticated, ancient and modern maize are partially buffered against the effects of geographic migration --- from a Mexican soil associated with ancestral maize, to a Canadian soil associated with modern hybrid agriculture. These results have implications for understanding the effects of domestication, migration, ex situ seed conservation and modern breeding, on the microbiome of one of the worlds most important food crops.

Chemical Inhibitors Suggest Endophytic Fungal Paclitaxel Is Derived from Both Mevalonate and Non-mevalonate-like Pathways

Taxus trees possess fungal endophytes reported to produce paclitaxel. Inhibitors that block early steps in plant paclitaxel biosynthesis were applied to a paclitaxel-producing fungus to determine whether these steps are shared. The plant paclitaxel backbone is reportedly derived from the non-mevalonate terpenoid pathway, while the side chain is phenylalanine-derived. Evidence that the shikimate pathway contributes to fungal paclitaxel was shown by decreased paclitaxel accumulation following inhibition of phenylalanine ammonia lyase. Expression of another shikimate pathway enzyme, 3-dehydroquinate synthase, coincided with paclitaxel production. The importance of the mevalonate pathway in fungal paclitaxel biosynthesis was shown by inhibition of fungal paclitaxel accumulation using compactin, a specific inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase. Expression of another mevalonate pathway enzyme, 3-hydroxy-3-methyl-glutaryl-CoA synthase, coincided with fungal paclitaxel accumulation. Unexpectedly, results from using fosmidomycin suggested that fungal paclitaxel requires 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), an enzyme in the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway normally found in bacteria/plants. Additional lines of evidence support this finding; first, a plant DXR antibody recognized a fungal peptide of the correct size; second, expression of an apparent fungal DXR ortholog correlated to changes in paclitaxel production; finally, BLAST searching identified a gene putatively encoding 1-deoxy-D-xylulose-5-phosphate synthase, the first enzyme in the MEP pathway in Aspergillus.

A fungal endophyte induces transcription of genes encoding a redundant fungicide pathway in its host plant

BackgroundTaxol is an anti-cancer drug harvested from Taxus trees, proposed ecologically to act as a fungicide. Taxus is host to fungal endophytes, defined as organisms that inhabit plants without causing disease. The Taxus endophytes have been shown to synthesize Taxol in vitro, providing Taxus with a second potential biosynthetic route for this protective metabolite. Taxol levels in plants vary 125-fold between individual trees, but the underlying reason has remained unknown.ResultsComparing Taxus trees or branches within a tree, correlations were observed between Taxol content, and quantity of its resident Taxol-producing endophyte, Paraconiothyrium SSM001. Depletion of fungal endophyte in planta by fungicide reduced plant Taxol accumulation. Fungicide treatment of intact plants caused concomitant decreases in transcript and/or protein levels corresponding to two critical genes required for plant Taxol biosynthesis. Taxol showed fungicidal activity against fungal pathogens of conifer wood, the natural habitat of the Taxol-producing endophyte. Consistent with other Taxol-producing endophytes, SSM001 was resistant to Taxol.ConclusionsThese results suggest that the variation in Taxol content between intact Taxus plants and/or tissues is at least in part caused by varying degrees of transcriptional elicitation of plant Taxol biosynthetic genes by its Taxol-producing endophyte. As Taxol is a fungicide, and the endophyte is resistant to Taxol, we discuss how this endophyte strategy may be to prevent colonization by its fungal competitors but at minimal metabolic cost to itself.