Field Inoculation Research Articles

Marcel van der Heijden.

Marcel van der Heijden.

Genetic diversity and symbiotic effectiveness of Phaseolus vulgaris-nodulating rhizobia in Kenya

Phaseolus vulgaris (common bean) was introduced to Kenya several centuries ago but the rhizobia that nodulate it in the country remain poorly characterised. To address this gap in knowledge, 178 isolates recovered from the root nodules of P. vulgaris cultivated in Kenya were genotyped stepwise by the analysis of genomic DNA fingerprints, PCR-RFLP and 16S rRNA, atpD, recA and nodC gene sequences. Results indicated that P. vulgaris in Kenya is nodulated by at least six Rhizobium genospecies, with most of the isolates belonging to Rhizobium phaseoli and a possibly novel Rhizobium species. Infrequently, isolates belonged to Rhizobium paranaense, Rhizobium leucaenae, Rhizobium sophoriradicis and Rhizobium aegyptiacum. Despite considerable core-gene heterogeneity among the isolates, only four nodC gene alleles were observed indicating conservation within this gene. Testing of the capacity of the isolates to fix nitrogen (N2) in symbiosis with P. vulgaris revealed wide variations in effectiveness, with ten isolates comparable to Rhizobium tropici CIAT 899, a commercial inoculant strain for P. vulgaris. In addition to unveiling effective native rhizobial strains with potential as inoculants in Kenya, this study demonstrated that Kenyan soils harbour diverse P. vulgaris-nodulating rhizobia, some of which formed phylogenetic clusters distinct from known lineages. The native rhizobia differed by site, suggesting that field inoculation of P. vulgaris may need to be locally optimised.

Field Inoculation and Classification of Maize Ear Rot Caused by Fusarium verticillioides.

Maize ear rot is a worldwide fungal disease mainly caused by Fusarium verticillioides and Fusarium graminearum. Maize planted in the field was inoculated with Fusarium verticillioides at the filling stage, 15 days after pollination. Two milliliters of spore suspension with a concentration of 5 x 106/ml was injected into the middle of the top ear using pricking ear method to cause maize ear rot. The thirty days after inoculation was the most suitable time for phenotypic evaluation of Fusarium resistance.

Large Scale Field Inoculation and Scoring of Maize Southern LeafBlight and Other Maize Foliar Fungal Diseases.

Field-grown maize is inoculated with Cochliobolus heterostrophus, causal agent of southern leaf blight disease, by dropping sorghum grains infested with the fungus into the whorl of each maize plant at an early stage of growth. The initial lesions produce secondary inoculum that is dispersed by wind and rain, causing multiple cycles of infection that assures a high uniform disease pressure over the entire field by the time of disease scoring, which occurs after anthesis. This method, with slight modifications, can also be used to study the maize fungal diseases northern leaf blight (caused by Exserohilum turcicum) and gray leaf spot (Cercospora zeae-maydis).

Research Article Selection of resistance sources to common bean anthracnose by field phenotyping and DNA marker-assisted screening

The main goal of this work was to select resistance sources to common bean anthracnose by field phenotyping and DNA marker-assisted screening. Fifty-five common bean genotypes, including differential varieties, characterized resistance sources, elite lines, cultivars and controls, were evaluated in a field inoculation trial and screened with SCAR markers linked to resistance genes that are important in Brazil. The field trial was carried out in Santo Antonio de Goias, GO, Brazil, during the fall/winter growing season of 2014, using artificial inoculation with a mixture of six races of Colletotrichum lindemuthianum, selected based on their high virulence and prevalence in Brazil. Amplification reactions with the SCAR markers previously identified as linked to important anthracnose resistance genes on Brazil followed standard procedures. Twenty-eight of the 58 genotypes were resistant to anthracnose (mean severity score ≤ 3.5). Ten of these 28 resistant genotypes stood out because they presented a mean anthracnose severity score of 1.0. Four of the six SCAR markers tested shown to be useful for the assisted selection of their respective target genes (SH18 and SAS13 for Co-42, SAB03 for Co-5, and SAZ20 for Co-6). Two carioca seeded elite lines were highlighted by the phenotypic and molecular screening: K10 (Co-34, Co-42, Co-5 and Co-6) and K13 (Co-4²). The phenotypic and molecular characterization of candidate resistance sources to common bean anthracnose based on their disease reaction in field inoculation trials and on the analysis with molecular markers linked to resistance genes has shown to be a useful strategy. These results aid in the selection of donor parents and resistant lines to be preferably explored by common bean breeding programs in Brazil.

Stable, fertile somatic hybrids between Sinapis alba and Brassica juncea show resistance to Alternaria brassicae and heat stress

Wild relatives of Brassica are a rich reservoir of genes that are invaluable for the improvement of cultivated species. Sinapis alba is a close relative of crop Brassicas that possesses several desirable traits such as tolerance to Alternaria black spot disease, heat stress, insect pests and nematodes. This study is aimed at developing and characterizing hybrids between Brassica juncea and S. alba with the ultimate goal of transferring genes for tolerance to Alternaria brassicae and heat stress, the traits that are lacking in cultivated Brassica. We generated three hybrids between B. juncea and S. alba through protoplast fusion. The hybridity was confirmed through cytology and molecular markers. While two of the hybrids were symmetric, the third one was asymmetric and had greater resemblance to B. juncea. Hybrids showed some characteristic features of the parents and were fully male and female fertile and also set seeds upon back crossing with the parent species. In vitro leaf assay and field inoculation studies revealed that the hybrids are highly resistant to A. brassicae. Besides, hybrids set seeds at temperature of > 38 °C when parents failed to produce seeds indicating that hybrids possess heat tolerance. These stable hybrids provide a reliable genetic resource for transfer of genes from S. alba into cultivated Brassica species.

Combined Field Inoculations of Pseudomonas Bacteria, Arbuscular Mycorrhizal Fungi, and Entomopathogenic Nematodes and their Effects on Wheat Performance.

In agricultural ecosystems, pest insects, pathogens, and reduced soil fertility pose major challenges to crop productivity and are responsible for significant yield losses worldwide. Management of belowground pests and diseases remains particularly challenging due to the complex nature of the soil and the limited reach of conventional agrochemicals. Boosting the presence of beneficial rhizosphere organisms is a potentially sustainable alternative and may help to optimize crop health and productivity. Field application of single beneficial soil organisms has shown satisfactory results under optimal conditions. This might be further enhanced by combining multiple beneficial soil organisms, but this remains poorly investigated. Here, we inoculated wheat plots with combinations of three beneficial soil organisms that have different rhizosphere functions and studied their effects on crop performance. Plant beneficial Pseudomonas bacteria, arbuscular mycorrhizal fungi (AMF), and entomopathogenic nematodes (EPN), were inoculated individually or in combinations at seeding, and their effects on plant performance were evaluated throughout the season. We used traditional and molecular identification tools to monitor their persistence over the cropping season in augmented and control treatments, and to estimate the possible displacement of native populations. In three separate trials, beneficial soil organisms were successfully introduced into the native populations and readily survived the field conditions. Various Pseudomonas, mycorrhiza, and nematode treatments improved plant health and productivity, while their combinations provided no significant additive or synergistic benefits compared to when applied alone. EPN application temporarily displaced some of the native EPN, but had no significant long-term effect on the associated food web. The strongest positive effect on wheat survival was observed for Pseudomonas and AMF during a season with heavy natural infestation by the frit fly, Oscinella frit, a major pest of cereals. Hence, beneficial impacts differed between the beneficial soil organisms and were most evident for plants under biotic stress. Overall, our findings indicate that in wheat production under the test conditions the three beneficial soil organisms can establish nicely and are compatible, but their combined application provides no additional benefits. Further studies are required, also in other cropping systems, to fine-tune the functional interactions among beneficial soil organisms, crops, and the environment.

Visual assessment and computer–assisted image analysis of Fusarium head blight in the field to predict mycotoxin accumulation in wheat grains

Phenotypic traits are regularly used to diagnose the development of Fusarium head blight (FHB) in the field, whereas mycotoxin accumulation in wheat grains can only be accurately evaluated through costly methods, such as high–performance liquid chromatography (HPLC). The aim of this study was to determine whether: (i) the results provided by existing commercial decision support tools could be anticipated using phenotypic measurements, including a novel technique of computer–assisted image analysis of spikes; and (ii) these measurements could avoid using HPLC. We monitored the FHB development during two consecutive years in highly contaminated plots in the Burgundy region (France). Contamination by crop residues was simulated through a field inoculation with barley grains artificially colonized by Fusarium graminearum. The development of the disease on spikes and harvested grains was assessed on one tolerant and two susceptible wheat varieties. The accumulated amounts of mycotoxins were measured in harvested grains using HPLC. As expected, the measured traits revealed that the inoculum responsible for infection on spikes mainly came from residues left on the soil surface, and the susceptible varieties were more diseased than the tolerant variety. Weather conditions had a strong effect on disease development. The novel computer–assisted image analysis technique had a better prediction power of deoxynivalenol accumulation, was more objective and time–saving than classical visual symptom assessments. This assessment method could be suitable to supplement the use of existing prediction tools and might avoid systematic and costly mycotoxin measurements in likely infected plots.

Does dormancy protect seeds against attack by the pathogenic fungus Fusarium tricinctum in a semiarid grassland of Northwest China?

Soil fungal pathogens can result in the failure of seedling establishment, but the effects of fungicide applications on seed/seedling survival have differed among studies. We assumed that the variation may relate to seed dormancy/germination characteristics and hypothesized that nondormant germinating seeds are more likely to be killed by fungal pathogens than dormant seeds. Dormant and nondormant seeds of Stipa bungeana and Lespedeza davurica were inoculated with a pathogenic fungus Fusarium tricinctum under laboratory and field conditions. The outcomes of seed/seedling fate and other parameters were evaluated. In the laboratory, nondormant seeds inoculated with F. tricinctum developed white tufts of mycelium on the radicles of germinating seeds causing them to quickly die, but dormant seeds remained intact. In contrast, in the field inoculation with F. tricinctum did not cause higher mortality of nondormant than dormant seeds but resulted in higher percentages of seedling death before they emerged from soil than the controls. Our results suggest that dormancy protects seeds from being attacked by some pathogens by preventing germination, but the protection is lost once germination has commenced. Further study involving various plant species with more seeds is needed to assess the generality of this pathogen-seed interaction hypothesis.

Biological Control of Cryphonectria parasitica in Romanian Protected Sweet Chestnut Forests

Asiatic fungus Cryphonectria parasitica has gradually infected all chestnut habitats, forest and horticulture plantations from the most important natural centres of sweet chestnut distribution in Romania. The objectives of this work were to understand the destruction rate of chestnut habitats under C. parasitica pressure, and to test the efficacy of pathogen control in revitalizing these protected forests. Successive inventories of forest health status were carried out in forest districts of Maramures and Gorj counties. C. parasitica biologic control using CHV1 virus has been tested. The methodology includes standard laboratory and field work techniques: local hipervirulent strain identification and conversion to hipovirulence, field canker inoculation, and treatment efficacy evaluation. The fungus killed all mature chestnut trees in roughly two decades. Field inoculation has been successful on chestnut and sessile oak (fungus secondary host) in experimental plots from both target zones. This is the first time in Romania when a forest habitat is recovered by means of biological control, after a severe dieback of the old forest caused by a lethal invasive pathogen.

Strong increase of durum wheat iron and zinc content by field-inoculation with arbuscular mycorrhizal fungi at different soil nitrogen availabilities

Since actual production of wheat often leads to human Fe and Zn deficiency, a better understanding of the potential of arbuscular mycorrhizal fungal (AMF) inoculation for micro-nutrient uptake of durum wheat is needed. Effects of AMF field inoculation and N availability were evaluated on an old and a modern durum wheat variety Following AMF inoculation, the modern variety showed a higher increase of the early root colonization respect to the old one, whereas at maturity root colonization was decreased by N fertilization. In the old variety grain N concentration was increased by inoculation when plants were not fertilized and at the 40–0-40 N, whereas in the modern variety inoculation did not change N concentration. By contrast, in AMF inoculated plots the modern variety showed a higher increase of Fe and Zn in grain compared to the old variety. Accordingly, at harvest, the modern variety showed an higher increase of a molecular operational taxonomic unit affiliated to Rhizophagus compared to the old variety. The inoculated isolate is a good durum wheat colonizer and the modern variety showed higher responsiveness to inoculation in terms of N, Fe and Zn grain concentration respect to the old one.

Evaluation of Resistance to Sheath Blight (Rhizoctonia solani Kuhn) by Field Inoculation Test and Improvement of the Resistance by Selection in Sorghum

Evaluation of Resistance to Sheath Blight (Rhizoctonia solani Kuhn) by Field Inoculation Test and Improvement of the Resistance by Selection in Sorghum

Interspecific interactions between the Sudden Oak Death pathogen Phytophthora ramorum and two sympatric Phytophthora species in varying ecological conditions

Abstract Even when introduced invasive pathogens lack their natural predators or competitors, they must still interact with other organisms in their introduced range. Sudden Oak Death (SOD), caused by Phytophthora ramorum (Oomycota), is an introduced disease causing large-scale tree mortality. Two additional Phytophthora species, Phytophthora nemorosa and Phytophthora pseudosyringae, cause significantly lower oak mortality, yet they also commonly colonize leaves of Umbellularia californica, the major transmissive host of SOD in California. We combined field surveys and inoculation experiments to understand disease prevalence dynamics and competitive interactions among these pathogen species. Despite the broader geographic distribution of P. nemorosa with respect to that of P. ramorum, our results suggest that P. nemorosa exhibits a narrower ecological amplitude and, in any given region, occupies fewer sites than P. ramorum. Our results additionally suggest that, perhaps due to priority effects, P. nemorosa can persist at levels comparable to those of P. ramorum in ecologically suitable plots when climate favors P. ramorum dormancy. However, P. ramorum prevalence increases to levels higher than those of the competing species when abundant rainfall triggers its sporulation. Understanding the determinants and outcomes of competition between these species has important implications for understanding the epidemiology and possible control strategies for Sudden Oak Death.

Natural distribution of Verticillium wilt on invasive Ailanthus altissima in eastern Austria and its potential for biocontrol

SummaryAilanthus altissima is a highly invasive tree species that has become established in warmer climatic zones of Europe. Verticillium wilt shows promise for biocontrol of this invasive tree species. In this study, we conducted a survey on the natural distribution of Verticillium species on A. altissima in eastern Austria. Results of a survey, across the range of A. altissima in Austria, indicated a widespread distribution of V. dahliae (56 of 77 sampled sites) and a rare occurrence of V. nonalfalfae (two of 77 sites). All sites positive for Verticillium spp. were situated at 130 m to 240 m above sea level. In addition, pathogenicity and virulence of an Austrian isolate of V. nonalfalfae, which is considered the most effective Verticillium species as biocontrol agent against A. altissima, were tested in field inoculations studies. Results revealed that disease progression on A. altissima is negatively influenced by high temperatures in summer. Therefore, inoculation in spring turned out to be the most effective to combat A. altissima. However, due to the common occurrence of V. dahliae in Austria, this pathogen might be considered as alternative biocontrol agent of A. altissima on sites in warmer climates. For that purpose, further investigations of the host range and non‐target effects of selected V. dahliae strains should be carried out.

Life cycle of Aecidium klugkistianum on Ligustrum and its new combination, Puccinia klugkistiana

During the survey of rust fungi in Jilin Province, China, we found Ligustrum obtusifolium (Oleaceae) heavily infected with a rust fungus, Aecidium klugkistianum in the Changchun area. The life cycle of A. klugkistianum was elucidated via field observations and inoculation experiments. This rust fungus produces uredinial and telial stages on Cleistogenes hackelii (Gramineae). These stages morphologically belong to Puccinia and are different from other fungi on Cleistogenes in terms of the size of the urediniospores. The new combination, P. klugkistiana is proposed for this rust fungus based on aecial stage name, A. klugkistianum. Its epitype specimen is designated.

Fruit quality of seedless watermelon grafted onto squash rootstocks under different production systems.

The market demand for seedless watermelon has been continuously increasing because of consumer preference. Grafting is a useful tool to manage soilborne diseases in watermelon production, but the use of squash rootstocks may negatively affect watermelon fruit quality. Currently, most research has focused on seeded cultivars, while grafting effects on seedless watermelons remain largely unknown. This multi-season study was conducted to assess the effects of squash rootstocks, including both Cucurbita maxima × C. moschata and C. moschata cultivars, with intact or excised and regenerated roots, on fruit quality of seedless watermelon 'Melody' using both instrumental and sensory measurements under different production scenarios. The grafted watermelon plants were also challenged by field inoculation with Fusarium oxysporum f.sp. niveum. A combination of instrumental measurements and consumer sensory analyses suggested that fruit quality of the seedless watermelon 'Melody' was not impacted by the use of the squash rootstocks used in this study, which included soluble solids content, titratable acidity, pH and most fruit sensory properties. Watermelon flesh firmness was increased by grafting but the grafting effect on lycopene content was inconclusive. Root excision and regeneration did not influence the grafting effect, whereas the grafting effect on flesh firmness varied among the rootstocks under Fusarium inoculation. Overall, grafting with squash rootstocks did not reduce fruit quality attributes of 'Melody' but improved texture. Our results support incorporating grafting into integrated management programs for seedless watermelon production. © 2017 Society of Chemical Industry.

Notes on rust fungi in China 3. Puccinia adenocauli comb. nov. and its life cycle and new host

The heteroecious and macrocyclic life cycle of Aecidium adenocauli was proved with field observations and inoculation experiments in China. Its spermogonial and aecial stages were produced on Adenocaulon himalaicum, and uredinial and telial stages were produced on Carex onoei, which is a new host for this rust fungus. Uredinial and telial stages (teleomorph) of this rust fungus were formerly described as Puccinia carici-adenocauli from Japan. However, the current International Code of Nomenclature (ICN (Melbourne)) has abolished the distinction between "anamorph" names and "teleomorph" names, and we propose the new combination Puccinia adenocauli, based on Ae. adenocauli 1913, which now has priority over P. carici-adenocauli 1999. Its neotype and epitype are also designated.

Cyclopiazonic Acid Is a Pathogenicity Factor for Aspergillus flavus and a Promising Target for Screening Germplasm for Ear Rot Resistance.

Aspergillus flavus, an opportunistic pathogen, contaminates maize and other key crops with carcinogenic aflatoxins (AFs). Besides AFs, A. flavus makes many more secondary metabolites (SMs) whose toxicity in insects or vertebrates has been studied. However, the role of SMs in the invasion of plant hosts by A. flavus remains to be investigated. Cyclopiazonic acid (CPA), a neurotoxic SM made by A. flavus, is a nanomolar inhibitor of endoplasmic reticulum calcium ATPases (ECAs) and a potent inducer of cell death in plants. We hypothesized that CPA, by virtue of its cytotoxicity, may serve as a key pathogenicity factor that kills plant cells and supports the saprophytic life style of the fungus while compromising the host defense response. This proposal was tested by two complementary approaches. A comparison of CPA levels among A. flavus isolates indicated that CPA may be a determinant of niche adaptation, i.e., isolates that colonize maize make more CPA than those restricted only to the soil. Further, mutants in the CPA biosynthetic pathway are less virulent in causing ear rot than their wild-type parent in field inoculation assays. Additionally, genes encoding ECAs are expressed in developing maize seeds and are induced by A. flavus infection. Building on these results, we developed a seedling assay in which maize roots were exposed to CPA, and cell death was measured as Evans Blue uptake. Among >40 maize inbreds screened for CPA tolerance, inbreds with proven susceptibility to ear rot were also highly CPA sensitive. The publicly available data on resistance to silk colonization or AF contamination for many of the lines was also broadly correlated with their CPA sensitivity. In summary, our studies show that i) CPA serves as a key pathogenicity factor that enables the saprophytic life style of A. flavus and ii) maize inbreds are diverse in their tolerance to CPA. Taking advantage of this natural variation, we are currently pursuing both genome-wide and candidate gene approaches to identify novel components of maize resistance to Aspergillus ear rot.

Host-plant variety and not climate determines the establishment and performance of Aceria lantanae (Eriophyidae), a biological control agent of Lantana camara in South Africa.

The flower-galling mite Aceria lantanae (Cook) (Trombidiformes: Eriophyidae) was released for the biological control of Lantana camara L. (Verbenaceae) in South Africa in 2007, but has displayed variable and patchy establishment throughout the weed's range. Surveys were undertaken in 2013-2014, both seasonally and during the mite's peak infestation periods, to determine the influence of climatic factors on its performance. Although there were seasonal differences in the percentages of mite-infested inflorescences, these did not differ significantly between altitudinal zones. There were also no significant relationships between the percentages of mite-infested inflorescences and either of annual rainfall, temperature or relative humidity. A field inoculation trial revealed significant differences between 10 common South African L. camara varieties in their susceptibility to A. lantanae. Only three varieties displayed appreciable susceptibility (50-61% of inflorescences infested), whereas six displayed only slight to moderate susceptibility (8-21%) and one displayed a lack of susceptibility (no infestation). These data support the contention that differential varietal susceptibility and not climate is responsible for the variable performance of A. lantanae on L. camara in South Africa. Complementing the current biotype of A. lantanae, originally sourced from Florida (USA), with other biotypes from different L. camara genotypes in Central and South America could increase the mite's impact on the weed.

Enhancement of ecosystem services during endophyte-assisted aided phytostabilization of metal contaminated mine soil

Endophytic plant growth-promoting bacteria (endophytes) were isolated from a variety of (pseudo)metallophytes growing in an abandoned Zn/Pb mine and then characterized according to their plant growth-promoting traits (i.e. ACC deaminase activity, IAA production, siderophore production, phosphate solubilising capacity, metal and salt tolerance and phenotypic characterization). Initially, under growth chamber conditions, an endophyte-assisted aided phytostabilization study was carried out with Festuca rubra plants (native vs. commercial variety) inoculated with a Pseudomonas sp. isolate and cow slurry as organic amendment. The effect of treatments on soil physicochemical and microbial indicators of soil quality, as well as plant physiological parameters and metal concentrations, was assessed. We performed a complementary interpretation of our data through their grouping within a set of ecosystem services. Although the application of cow slurry had the most pronounced effects on soil quality indicators and ecosystem services, the growth of native F. rubra plants reduced soil bioavailability of Cd and Zn by 19 and 22%, respectively, and enhanced several soil microbial parameters. On the other hand, endophyte (Pseudomonas sp.) inoculation improved the physiological status of F. rubra plants by increasing the content of carotenoids, chlorophylls and Fv/Fm by 69, 65 and 37%, respectively, while also increasing the values of several soil microbial parameters. Finally, a consortium of five endophyte isolates was used for an endophyte-assisted aided phytostabilization field experiment, where lower metal concentrations in native excluder plants were found. Nonetheless, the field inoculation of the endophyte consortium had no effect on the biomass of native plants.