Novel Trichoderma Isolates Alleviate Water Deficit Stress in Susceptible Tomato Genotypes.

Symbiotic fungi in the genus Trichoderma can induce abiotic stress tolerance in crops. The beneficial effects of Trichoderma on water deficit stress are poorly understood and may be isolate-specific. Our objective was to evaluate a collection of Nepalese Trichoderma isolates and their efficacy to improve tomato (Solanum lycopersicum) growth under water deficit. Variable growth in low moisture environments was observed among Trichoderma isolates from Nepal, Ohio, and commercial sources using in vitro assays. The overall performance of the population decreased when cultured under conditions of decreasing matric water potential (0.0, -2.8, -4.8, and -8.5 Ψ). Twelve isolates were selected for evaluation for their potential to elicit drought tolerance in greenhouse-grown 'Roma Organic' tomatoes. Plants treated with T. asperelloides-NT33 had higher shoot weight than the non-inoculated control (T0) under water deficit stress conditions. Further, the stress-reducing efficacy of isolates T. asperelloides-NT33, T. asperellum-NT16, T. asperelloides-NT3, and commercial T. harzianum-T22 were tested on tomato genotypes with differing tolerance to drought ['Roma Organic,' 'Jaune Flamme,' and 'Punta Banda']. The water deficit susceptible genotypes 'Roma Organic' and 'Jaune Flamme' inoculated with isolate NT33 had significantly higher shoot weight (37 and 30% respectively; p < 0.05) compared to the non-inoculated control under water deficit stress conditions. In drought tolerant 'Punta Banda,' shoot weight was also significantly greater in NT33 inoculated plants under water deficit stress conditions, but with lower magnitude difference (8%; p < 0.05). Our results demonstrate differences in the ability of Trichoderma isolates to confer tolerance to water deficit in tomato with NT33 potentially relieving stress. Tomato genotypes also play a role in the outcome of interactions with the Trichoderma isolates we tested.

Diverging landscape impacts on macronutrient status despite overlapping diets in managed (Apis mellifera) and native (Melissodes desponsa) bees.

Declining pollinator populations worldwide are attributed to multiple stressors, including the loss of quality forage. Habitat management in agricultural areas often targets honey bees (Apis mellifera L.) specifically, with the assumption that native bees will benefit from an 'umbrella species' strategy. We tested this theory using a conservation physiology approach to compare the effects of landscape composition and floral dietary composition on the physiological status of honey bees and Melissodes desponsa in eastern South Dakota, USA. The total glycogen, lipid and protein concentrations were quantified from field collected bees. Next-generation sequencing of the trnL chloroplast gene from bee guts was used to evaluate dietary composition. The effects of landscape and dietary composition on macronutrient concentrations were compared between bee species. As the mean land-use patch area increased, honey bee glycogen levels increased, though M. desponsa experienced a decrease in glycogen. Protein levels decreased in honey bees as the largest patch index, a measure of single patch dominance, increased versus M. desponsa. Lipids in both species were unaffected by the measured landscape variables. Dietary analysis revealed that honey bees foraged preferentially on weedy non-native plant species, while M. desponsa sought out native and rarer species, in addition to utilizing non-native plants. Both species foraged on Asteraceae, Oleaceae and Fabaceae, specifically Melilotus sp. and Medicago sp. Dietary composition was not predictive of the macronutrients measured for either species. Together, these data highlight the management importance of including patch area in conservation recommendations, as bee species may have divergent physiological responses to landscape characteristics. While solitary bees may forage on weedy introduced plants in agricultural areas, robust strategies should also reincorporate native plant species, though they may not be preferred by honey bees, to maximize overall health and diversity of pollinator communities.

Previous crop and rotation history effects on maize seedling health and associated rhizosphere microbiome.

To evaluate crop rotation effects on maize seedling performance and its associated microbiome, maize plants were grown in the greenhouse in soils preceded by either maize, pea, soybean or sunflower. Soils originated from a replicated field experiment evaluating different four-year rotation combinations. In the greenhouse, a stressor was introduced by soil infestation with western corn rootworm (WCR) or Fusarium graminearum. Under non-infested conditions, maize seedlings grown in soils preceded by sunflower or pea had greater vigor. Stress with WCR or F. graminearum resulted in significant root damage. WCR root damage was equivalent for seedlings regardless of soil provenance; whereas F. graminearum root damage was significantly lower in maize grown in soils preceded by sunflower. Infestation with WCR affected specific microbial taxa (Acinetobacter, Smaragdicoccus, Aeromicrobium, Actinomucor). Similarly, F. graminearum affected fungal endophytes including Trichoderma and Endogone. In contrast to the biological stressors, rotation sequence had a greater effect on rhizosphere microbiome composition, with larger effects observed for fungi compared to bacteria. In particular, relative abundance of Glomeromycota was significantly higher in soils preceded by sunflower or maize. Defining the microbial players involved in crop rotational effects in maize will promote selection and adoption of favorable crop rotation sequences.

Pathogen regulation of plant diversity via effective specialization.

The Janzen-Connell (JC) hypothesis, one of the most influential hypotheses explaining forest diversity, is inconsistent with evidence that tree species share the same natural enemies. Through the discussion of seedling diseases from a pathogen-centered perspective, we expand the JC hypothesis to tie in host-pathogen-environment interactions at three levels: local adaptation, host specificity of the combined effect of multiple infections, and environmental modulation of disease. We present evidence from plant pathology, disease ecology, and host-parasite evolution relevant to (but not commonly associated with) forest species diversity maintenance. This expanded view of the JC hypothesis suggests ways to direct new experiments to integrate research on pathogen local adaptation, co-infection, and environmental effects on infection by using high-throughput molecular techniques and statistical models.

Linking sequence to function in soil bacteria: sequence-directed isolation of novel bacteria contributing to soilborne plant disease suppression.

Microbial community profiling of samples differing in a specific ecological function, i.e., soilborne plant disease suppression, can be used to mark, recover, and ultimately identify the bacteria responsible for that specific function. Previously, several terminal restriction fragments (TRF) of 16S rRNA genes were statistically associated with damping-off disease suppression. This work presents the development of sequence-based TRF length polymorphism (T-RFLP)-derived molecular markers to direct the identification and isolation of novel bacteria involved in damping-off pathogen suppression. Multiple sequences matching TRF M139 and M141 were cloned and displayed identity to multiple database entries in the genera incertae sedis of the Burkholderiales. Sequences matching TRF M148, in contrast, displayed greater sequence diversity. A sequence-directed culturing strategy was developed using M139- and M141-derived markers and media reported to be selective for the genera identified within this group. Using this approach, we isolated and identified novel Mitsuaria and Burkholderia species with high levels of sequence similarity to the targeted M139 and M141 TRF, respectively. As predicted, these Mitsuaria and Burkholderia isolates displayed the targeted function by reducing fungal and oomycete plant pathogen growth in vitro and reducing disease severity in infected tomato and soybean seedlings. This work represents the first successful example of the use of T-RFLP-derived markers to direct the isolation of microbes with pathogen-suppressing activities, and it establishes the power of low-cost molecular screening to identify and direct the recovery of functionally important microbes, such as these novel biocontrol strains.

Field management effects on damping-off and early season vigor of crops in a transitional organic cropping system.

Transitioning farmland to certified organic vegetable production can take many paths, each varying in their costs and benefits. Here, the effects of four organic transition strategies (i.e., tilled fallowing, mixed-species hay, low-intensity vegetables, and intensive vegetable production under high tunnels), each with and without annual compost applications for 3 years prior to assessment, were characterized. Although transition cropping strategies differed in soil chemistry (P < 0.05), the magnitude of the changes typically were marginal and pairwise comparisons were rarely significant. In contrast, the compost amendment had a much greater impact on soil chemistry regardless of cropping strategy. For example, percent C and total P increased by 2- to 5-fold and K increased from 6- to 12-fold. Under controlled conditions, damping-off of both edamame soybean (cv. Sayamusume) and tomato (cv. Tiny Tim) was reduced from 2 to 30% in soils from the mixed-hay transition. In the field, damping-off of both crops was also significantly lower in plots previously cropped to hay (P < 0.05). Although not always significant (P < 0.05), this pattern of suppression was observed in all four of the soybean experiments and three of the four tomato experiments independent of compost application. The compost amendments alone did not consistently suppress damping-off. However, plant height, fresh weight, and leaf area index of the surviving seedlings of both crops were greater in the compost-amended soils regardless of the transitional cropping treatment used (P < 0.05 for most comparisons). These data indicate that mixed-hay cropping during the transition periods can enhance soil suppressiveness to damping-off. In addition, although compost amendments applied during transition can improve crop vigor by significantly enhancing soil fertility, their effects on soilborne diseases are not yet predictable when transitioning to certified organic production.

Comparative analysis of Xenopus VegT, the meso-endodermal determinant, identifies an unusual conserved sequence.

The transcription factor, VegT, is the meso-endodermal determinant in Xenopus laevis. We examined VegT orthologs from several anuran amphibians and the urodele amphibian, the Mexican axolotl. In addition to the conserved T-box, the DNA-binding domain, the orthologs share a conserved 57 amino acid domain at the C-terminal. Most striking is a 17-nucleotide (nt) sequence near the 3' end of the open reading frame. The 17 nts are absolutely conserved among the anurans, whose last common ancestor lived 200 million years ago. As an initial test of the function of the 17 nts, 27 or 49 amino acids, which include the six amino acids coded by the 17 (+1) nts, were deleted from the C-terminal of VegT. These truncated VegT's retained some transcriptional activity, indicating that the 17 nts are not absolutely required for this function. The function of the highly conserved 17 nts is unknown. Two possibilities are that the conserved 17 nts interact with the cytoskeleton or that they are a target for regulation by a microRNA.

Farm Management Effects on Rhizosphere Colonization by Native Populations of 2,4-Diacetylphloroglucinol-Producing Pseudomonas spp. and Their Contributions to Crop Health.

ABSTRACT Analyses of multiple field experiments indicated that the incidence and relative abundance of root-colonizing phlD+ Pseudomonas spp. were influenced by crop rotation, tillage, organic amendments, and chemical seed treatments in subtle but reproducible ways. In no-till corn plots, 2-year rotations with soybean resulted in plants with approximately twofold fewer phlD+ pseudomonads per gram of root, but 3-year rotations with oat and hay led to population increases of the same magnitude. Interestingly, tillage inverted these observed effects of cropping sequence in two consecutive growing seasons, indicating a complex but reproducible interaction between rotation and tillage on the rhizosphere abundance of 2,4-diacetlyphloroglucinol (DAPG) producers. Amending conventionally managed sweet corn plots with dairy manure compost improved plant health and also increased the incidence of root colonization when compared with nonamended plots. Soil pH was negatively correlated to rhizosphere abundance of phlD+ pseudomonads in no-till and nonamended soils, with the exception of the continuous corn treatments. Chemical seed treatments intended to control fungal pathogens and insect pests on corn also led to more abundant populations of phlD in different tilled soils. However, increased root disease severity generally was associated with elevated levels of root colonization by phlD+ pseudomonads in no-till plots. Interestingly, within a cropping sequence treatment, correlations between the relative abundance of phlD and crop stand or yield were generally positive on corn, and the strength of those correlations was greater in plots experiencing more root disease pressure. In contrast, such correlations were generally negative in soybean, a difference that may be partially explained by difference in application of N fertilizers and soil pH. Our findings indicate that farming practices can alter the relative abundance and incidence of phlD+ pseudomonads in the rhizosphere and that practices that reduce root disease severity (i.e., rotation, tillage, and chemical seed treatment) are not universally linked to increased root colonization by DAPG-producers.

Development of the dendrobatid frog Colostethus machalilla.

To provide a developmental correlate with other frogs, we prepared a normal table of development for the dendrobatid, Colostethus machalilla and analyzed the morphology of its early development. This frog reproduces in captivity and deposits moderately sized eggs (1.6 mm in diameter) in terrestrial nests. The father guards the embryos until tadpole hatching. We divided development until hatching into 25 stages and implemented methods for in vitro culture of the embryos. The external and internal morphology of embryos were evaluated by observations in whole mount and in sections. Neural, notochord and somite specific antibodies were used to analyze gene expression patterns by immunostaining of embryos. Embryonic development of C. machalilla is slow and deviates from Xenopus laevis. In C. machalilla the elongation of the notochord, neural plate and somite formation occur after blastopore closure, possibly due to a delay in the dorsal convergence and extension movements. The gastrula of C. machalilla also deviates from X. laevis. The archenteron remains small until blastopore closure, where small cells accumulate at the blastopore lips. Simultaneously, the blastocoel roof thins until it becomes a monolayer of cells. Although C. machalilla does not form an embryonic disk, its thick blastopore lips resemble the embryonic disk of the marsupial frog Gastrotheca riobambae and represent an interesting deviation from the gastrulation pattern observed in X. laevis.

Expression of Brachyury during development of the dendrobatid frog Colostethus machalilla.

The expression of Brachyury (Bra) during development of Colostethus machalilla was analyzed with a polyclonal antibody. The observed molecular mass of Bra was of 48 kDa, as in Xenopus laevis. During cleavage, low levels of Bra were expressed. In contrast, in the blastula Bra became up-regulated, and Bra protein was present in a wide ring of surface cells. The surface expression of Bra disappeared in the gastrula, and a new ring of Bra-positive nuclei was detected in deep cells around the closing blastopore. The C. machalilla external and internal rings of Bra-positive nuclei apparently mark the prospective mesoderm in the blastula and gastrula, respectively. The two Bra expression rings were dissociated in time in the fairly slow developing embryos of this frog. Brachyury expression in the notochord became visible only after the blastopore closed, in contrast with X. laevis. In addition, Bra expression in the notochord indicated that dorsal convergence and extension occurred after blastopore closure. The C. machalilla Bra-positive notochord was originally exposed on the gastrocoel roof, in agreement with a superficial component of the prospective mesoderm.