Methods for Assessment of Viability and Germination of Plasmodiophora brassicae Resting Spores

Clubroot caused by the obligate biotrophic parasite Plasmodiophora brassicae is a destructive soil borne disease of cruciferous crops. Resting spores of P. brassicae can survive in the soil for a long period without hosts or external stimulants. The viability and germination rate of resting spores are crucial factors of the inoculum potential in the field. The accurate assessment of viability and germination rate is the foundation to evaluate the effect of control methods. In this study, we evaluated several methods for the assessment of viability and germination rate of P. brassicae resting spores. Dual staining with calcofluor white-propidium iodide (CFW-PI) or single stain with Evans blue showed reliable accuracy in estimating viability. CFW-PI was capable of reliably determining the viability within 10 min, while Evans blue required overnight incubation to obtain accurate results. Due to DNA degradation of heat treatments, acetone was selected to evaluate the efficiency of propidium monoazide (PMA)–quantitative PCR (qPCR) used for the quantification of DNA from viable cells. The staining with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI) and the use of differential interference contrast microscopy were suitable for the determination of resting spore germination rates. The latter method also allowed recording individual germination states of spores. Alternatively, dual staining with CFW-Nile red was successfully used to assess the germination rate of resting spores with a lethal pre-treatment. This study evaluates and confirms the suitability of various microscopic and molecular genetic methods for the determination of viability and germination of P. brassicae resting spores. Such methods are required to study factors in the soil regulating survival, dormancy and germination of P. brassicae resting spores causing clubroot disease in Brassicaceae hosts and therefore are fundamental to develop novel strategies of control.

Quantification of Phytophthora infestans population densities and their changes in potato field soil using real-time PCR

Tuber infection of Phytophthora infestans often occurs at harvest. However, it is difficult to accurately estimate the population densities of P. infestans in soil, especially Japanese soil. In the present study, P. infestans DNA was extracted from soil samples using a modified CTAB-bead method and quantified using real-time PCR to accurately, rapidly and easily estimate the P. infestans population densities in upland soils in Japan. P. infestans was well quantified in eleven types of soil samples, including nine types of upland soils in Japan, that were artificially inoculated with a zoosporangia suspension. The amounts of P. infestans DNA estimated by the real-time PCR were proportional to the inoculum densities. In the non-controlled experimental potato field, P. infestans population densities in soil corresponded to the development of symptoms and were correlated with the number of lesions on the potato foliage. These results imply that the proposed real-time PCR assay is suitable for the estimation or monitoring of P. infestans population densities in upland soils in Japan. The population densities at the ridge bottoms were larger than those at any other location in commercial potato fields. These results were similar to those of a previous report using a bioassay. Moreover, a correlation between DNA quantity and inoculum potential was observed. In conclusion, the real-time PCR assay developed in this study is suitable for indirect estimation of the inoculum potential of P. infestans.

Partial Retention of Legacy Trees Protect Mycorrhizal Inoculum Potential, Biodiversity, and Soil Resources While Promoting Natural Regeneration of Interior Douglas-Fir

Clearcutting reduces proximity to seed sources and mycorrhizal inoculum potential for regenerating seedlings. Partial retention of legacy trees and protection of refuge plants, as well as preservation of the forest floor, can maintain mycorrhizal networks that colonize germinants and improve nutrient supply. However, little is known of overstory retention levels that best protect mycorrhizal inoculum while also providing sufficient light and soil resources for seedling establishment. To quantify the effect of tree retention on seedling regeneration, refuge plants, and resource availability, we compared five harvesting methods with increasing retention of overstory trees (clearcutting (0% retention), seed tree (10% retention), 30% patch retention, 60% patch retention, and 100% retention in uncut controls) in an interior Douglas-fir-dominated forest in British Columbia. Regeneration increased with proximity to legacy trees in partially cut forests, with increasing densities of interior Douglas-fir, western redcedar, grand fir, and western hemlock seedlings with overstory tree retention. Clearcutting reduced cover of ectomycorrhizal refuge plants (from 80 to 5%) while promoting arbuscular mycorrhizal plants the year after harvest. Richness of shrubs, herbs, and mosses declined with increasing harvesting intensity, but tree richness remained at control levels. The presence of legacy trees in all partially cut treatments mitigated these losses. Light availability declined with increasing overstory cover and proximity to leave trees, but it still exceeded 1,000 W m−2 in the clearcut, seed tree and 30% retention treatments. Increasing harvesting intensity reduced aboveground and belowground C stocks, particularly in live trees and the forest floor, although forest floor losses were also substantial where thinning took place in the 60% retention treatment. The loss of forest floor carbon, along with understory plant richness with intense harvesting was likely associated with a loss of ectomycorrhizal inoculum potential. This study suggests that dispersed retention of overstory trees where seed trees are spaced ~10–20 m apart, and aggregated retention where openings are <60 m (2 tree-lengths) in width, will result in an optimal balance of seed source proximity, inoculum potential, and resource availability where seedling regeneration, plant biodiversity, and carbon stocks are protected.

Quantification and conceptual validation of the inoculum potential of Sclerotinia sclerotiorum in soybean and bean seeds

The fungus Sclerotinia sclerotiorum, the causal agent of white mold, is widespread throughout the world. The disease is considered to be one of the major diseases of soybean and bean crops in Brazil. The pathogen S. sclerotiorum is spread by soybean and bean seeds both in the form of sclerotia and dormant mycelium inside the seeds. The objective of this work was to evaluate the relationship between different potentials of S. sclerotiorum in soybean and bean seeds and the performance of these seeds, as well as to verify the localization and quantification of the inoculum of the pathogen in the seeds inoculated by Real-time PCR (qPCR), validating the term inoculum potential. Soybean and bean seeds were inoculated with the fungus by the osmotic conditioning method based on the exposure of the seeds to the fungus for periods of 24 h, 48 h, 72 h, and 96 h. Molecular analysis was carried out by means of qPCR in whole seeds and dissected in the integument, cotyledon and embryonic axis. The results showed that the effects of S. sclerotiorum on seed germination and vigor were progressive and proportional to the increases in inoculum potentials, since there was more severe damage to the seeds and consequently to the emerged plants at the highest potential (P96). The inoculum of the pathogen was found in all parts of the evaluated seeds, even at its lowest inoculum potential (P24), with an increasing DNA concentration, and the integument obtained a greater amount of DNA than the embryo, in comparison.

Effect of chemical treatment on the physiological and sanitary quality of stored coffee seeds

Maintaining the health of coffee seeds is especially important during storage, as soil fungi and storage fungi can considerably reduce seed quality. Thus, chemical treatments for protection of seeds in storage becomes important in agricultural production. It is necessary to evaluate the effects of these treatments on seedling development and the protection they provide against storage fungi, aiming at seed longevity and preventing rapid deterioration. The aim of this study was to evaluate the effect of chemical treatment on the physiological and sanitary quality of stored coffee seeds. Seeds of five Coffea arabica cultivars were pre-dried, treated with Vitavax®-Thiram, and placed in cold storage at 10 °C for nine months. Seed physiological quality was evaluated every three months by the germination test and by determination of root emergence percentage, seedlings with expanded cotyledonary leaves, and seedling dry matter. Seed health quality was assessed by the health test. The chemical treatment with Vitavax-Thiram does not affect the physiological quality of stored Coffea arabica seeds. Seed treatment before storage is effective in reducing the inoculum potential of Fusarium spp. and Phoma spp. in coffee seeds.

Studies into Fungal Decay of Wood In Ground Contact—Part 1: The Influence of Water-Holding Capacity, Moisture Content, and Temperature of Soil Substrates on Fungal Decay of Selected Timbers

This article presents the results from two separate studies investigating the decay of wood in ground contact using adapted versions of laboratory-based terrestrial microcosm (TMC) tests according to CEN/TS 15083-2:2005. The first study (A) sought to isolate the effect of soil water-holding capacity (WHCsoil [%]) and soil moisture content (MCsoil [%WHCsoil]) on the decay of five commercially important wood species; European beech (Fagus sylvatica), English oak heartwood (Quercus robur), Norway spruce (Picea abies), Douglas-fir heartwood (Pseudotsuga menziesii), and Scots pine sapwood (Pinus sylvestris), while keeping soil temperature (Tsoil) constant. Combinations of soil mixtures with WHCsoil of 30%, 60%, and 90%, and MCsoil of 30%, 70%, and 95%WHCsoil were utilized. A general trend showed higher wood decay, measured in oven-dry mass loss (MLwood [%]), for specimens of all species incubated in soils with WHCsoil of 60% and 90% compared to 30%. Furthermore, drier soils (MCsoil of 30 and 70%WHCsoil) showed higher MLwood compared to wetter soils (95%WHCsoil). The second study (B) built on the first’s findings, and sought to isolate the effect of Tsoil and MCsoil on the decay of European beech wood, while keeping WHCsoil constant. The study used constant incubation temperature intervals (Tsoil), 5–40 °C, and alternating intervals of 10/20, 10/30, and 20/30 °C. A general trend showed drier MCsoil (60%WHCsoil), and Tsoil of 20–40 °C, delivered high wood decay (MLwood > 20%). Higher MCsoil (90%WHCsoil) and Tsoil of 5–10 °C, delivered low wood decay (MLwood < 5%). Alternating Tsoil generally delivered less MLwood compared to their mean constant Tsoil counterparts (15, 20, 25 °C). The results suggest that differences in wood species and inoculum potential (WHCsoil) between sites, as well as changes in MCsoil and Tsoil attributed to daily and seasonal weather patterns can influence in-ground wood decay rate.

Fusarium and mycotoxin content of harvested grain was not related to tillage intensity in Norwegian spring wheat fields

To mitigate the risk of erosion and nutrient runoff, reduced tillage has become more prevalent in Norway. Within within recent decades, there have been some years with relatively high occurrence of Fusarium head blight and mycotoxins in Norwegian cereal grain. This is thought to have been caused by an increased inoculum potential (IP) of Fusarium spp. due to larger amount of crop residues remaining on the soil surface, in combination with weather conditions promoting fungal growth and infection of cereal plants. The objective of this work was to elucidate the influence of different tillage practices on the IP of Fusarium spp. and the subsequent Fusarium-infection and mycotoxin contamination of spring wheat grain at harvest. Tillage trials were conducted at two locations in southeast Norway (Solør and Toten) over three years, 2010-2012. Residues of wheat from the previous year were collected in spring. Fusarium avenaceum and Fusarium graminearum were the most common Fusarium species recorded on wheat straw residues. IP was calculated as the percentage of the residues infested with Fusarium spp. multiplied by the proportion of the soil surface covered with residues. The IP of Fusarium spp. was lower in ploughed plots compared to those tilled with harrowing only. Ploughing in spring resulted in a similarly low IP as autumn ploughing. In contrast, harrowing in autumn generally reduced IP more than did spring harrowing. The mycotoxin levels in the harvested wheat were generally low, except for deoxynivalenol at high levels in Solør 2011. Despite a lower IP of ploughed versus harrowed plots, this was not reflected in the content of Fusarium and mycotoxins in harvested grain. The Fusarium species that dominated in the residues examined in this study were the same as those detected in the harvested grain, supporting the finding that residues are an important source of inoculum.