Use of frozen bagged seed inoculum for secondary metabolite and bioconversion processes at the pilot scale

Frozen bagged seed inoculum was prepared, thawed and tested for seven cultures. Thawing techniques were developed and other key influences on thawing rate were quantified; seed bag thawing without a water bath rarely required more than 4 to 5 h and was as short as 0.5 to 1 h for lower fill volume bags. Testing included growth of bagged seed as a function of bag fill volume (0.5, 1.0, 2.0, and 3.5 L), comparison of culture age at time of bagging, growth of bagged versus laboratory-prepared seed, productivity of production cultures derived from bagged versus laboratory-prepared seed, growth of bagged seed as a function of volume percent glycerol added at time of bagging, and growth of bagged seed as a function of frozen storage time and temperature. For each culture tested, conditions were developed such that seed tanks inoculated with bagged seed showed only minimal delay in attaining the target oxygen uptake rate (OUR) relative to seed tanks inoculated with laboratory-prepared inoculum. Although the bag fill volume did influence culture growth in some cases, bag fill volumes required were reasonable (typically 2.0 to 3.5 L) compared with laboratory seed inoculum volumes of 2.0 L. In the most remarkable example, frozen bagged seed was prepared from a second-stage seed-tank cultivation of Glarca lozoyensis, then thawed and inoculated into first-stage seed medium. It grew to the desired OUR in a similar timeframe as laboratory-prepared inoculum inoculated into first-stage seed medium. Thus, the frozen bagged seed replaced an existing laboratory inoculum preparation period of 7 days without an appreciable delay in either of the two subsequent seed-tank growth stages. Furthermore, productivities were found to be comparable for bagged-seed-derived and laboratory-seed-derived production cultivations for four different fermentation processes.     

The role of seed-tuber and stem inoculum in the development of gangrene in potatoes

To determine the relative importance of seed tubers and stems as sources of inoculum for potato gangrene in progeny tubers, different levels of inoculum of Phoma exigua var. foveata were established in field experiments by planting rotting or contaminated seed tubers and by inoculating stems shortly before haulm desiccation. The pathogen was only occasionally detected by isolation from inside green stems in June and July on plants growing from contaminated but unrotted seed. The incidence of pycnidia on desiccated stems increased with increasing concentration of inoculum contaminating seed tubers and with increasing time interval between desiccation and harvest. Stem infection was probably derived from inoculum on seed tubers spreading via the soil to the stem bases. Stem inoculation of plants growing from uninoculated or uncontaminated seed greatly increased both the gangrene potential of progeny tubers (defined as % wounds with gangrene after uniformly damaging tubers and storing them at 5°C for 12 wk), and tuber contamination (defined as % wounds with gangrene after spreading tuber-borne soil onto test tuber slices and storing them at 5°C for 8 wk). However, when stems of plants growing from rotting or contaminated seed were cut at ground level and removed before desiccation, gangrene potential of progeny tubers was only slightly less than that of tubers from untreated plots. There was no evidence that soil inoculum or aerial spread played a significant role in disease development. Gangrene potential and contamination of progeny tubers were related to Contamination levels on seed tubers. Some transmission also occurred from rotted seed tubers to progeny. Inoculum levels around progeny tubers increased rapidly after desiccation even in plots where stems had previously been cut at soil level and removed to eliminate pycnidial development above ground as a source of inoculum. Both gangrene potential and contamination of progeny tubers were greater on cv. Ulster Sceptre than on cv. Pentland Crown. The results showed that the inoculum on seed tubers, whether from rots or surface contamination, contributed more to the contamination of progeny tubers at harvest than did the inoculum from pycnidia on stems following desiccation of the haulm.     

Relationship of seed source and arbuscular mycorrhizal fungi inoculum type to growth and colonization of big bluestem (Andropogon gerardii)

Big bluestem (Andropogon gerardii) is a key grass of tallgrass prairies and is commonly included in restoration projects. In many cases, it has been found to benefit significantly from arbuscular mycorrhizal (AM) fungi, however results have varied under non-sterile soil conditions. This study investigated the effects of two types of AM fungi inoculum (commercial and prairie) on growth and root colonization of big bluestem from five different seed sources grown in non-sterile soils. Seed sources were collected from five remnant prairies in the Tallgrass Prairie Peninsula located in the Midwestern United States. Growth of big bluestem and root length colonized by AM fungi was highly variable among seed source treatments. Overall, percentage of root length colonized by AM fungi was positively correlated with the total dry weight of plants, and plants that received inoculum generally grew better than those that did not receive inoculum. Inoculum treatment affected both big bluestem growth and percentage root length colonized and there was an interaction between seed source and inoculum treatment relative to colonization. Root colonization responses were not significantly different between the prairie and commercial inoculum types, although there was a significant response between plants that received additional inoculum as opposed to no additional inoculum. Seed sources from Ohio and Illinois had the highest biomasses and greatest percentage of root length colonized while plants from Wisconsin and Missouri grew poorly and had low root colonization. These results demonstrate the importance of considering both seed source and inoculum type before the incorporation of AM inoculum to prairie restoration projects.     

Epidemiology and strategy for the control of halo-blight of beans

A halo-blight epidemic model based on the equation of Van der Plank (1963) is described. The model has been used to determine tolerance levels for seed infection and to compare the effectiveness of chemical control measures based on foliar sprays and seed treatments.
Reduction of primary inoculum, derived from infected seed, by disease exclusion through seed testing, or by chemical seed treatment will give effective disease control under British conditions (i.e. with an expected apparent infection rate ( r ) of 0.15 per infected plant per day). Under climatic conditions more favourable for the disease where higher r values would occur, control treatments directed against primary inoculum only would be less effective. Under such conditions, it is predicted that chemical sprays, which have no effect on primary inoculum but cause a reduction in the rate of disease increase, would be more effective. The most effective disease control, however, would be a combination of treatments.     

Seed treatment technology: An attractive delivery system for controlling root parasitic weed Striga with mycoherbicide

Coating sorghum seeds with Fusarium oxysporum (Foxy 2) for control of the root parasitic weed Striga, appears to be an attractive option for minimizing the inoculum amount, establishing the biocontrol agent in the potential infection zone of the host plants, and offering a simple, easy and economical delivery system. Our investigations resulted in the selection of appropriate seed coating materials and a suitable type and form of fungal inoculum. The coating materials tested were arabic gum (AG10%, 20%, 40%), carboxymethylcellulose (CMC1%, 2%) and pectin (LS 440, LM-5 CS) 1%, while the fungal inoculum included microconidia and fresh and dried chlamydospores produced using different substrates. Foxy 2 survived the seed treatment processing and showed excellent viability on seeds for at least 8 months of storage after coating. In general, the performance of 40% arabic gum in combination with dried chlamydospores was the best among the other types of inoculum and coating material tested. Regardless of the type and form of inoculum and coating materials tested, Foxy 2 was able to colonize all roots, even root tips and hairs of the host (sorghum), thereby meeting important criteria of a promising candidate for controlling Striga when applied as a seed treatment. The efficacy of treated sorghum seed with Foxy 2 using different coating materials in reducing S. hermonthica infestation was evaluated in pot and root chamber trials. Foxy 2 markedly reduced Striga emergence and dry weight and increased the percentage of the diseased emerged Striga shoots. However, the efficacy of seed coating varied according to the type and form of fungal inoculum as well as coating material. Coating sorghum seed with dried chlamydospore inoculum homogenized into 20% arabic gum (as adhesive) showed the highest efficacy of 81 and 77% (i.e., percent reduction in healthy emerged Striga shoots compared to the control treatment) against Striga using either sterilized or non-sterilized soil, respectively. In root chamber bioassays, the application of Foxy 2 in combination with AG40% significantly caused disease in 77% of the germinated Striga seeds and in all tubercles after 25 days of sowing. These findings provide an optimized coating protocol as an attractive delivery system for bioherbicides for root parasitic weeds.     

The Seed-borne Inoculum of Peronospora manshurica, Causal Agent of Soybean Downy Mildew

The seed-borne inoculum of P. manshurica is described using various microscopic techniques and staining reactions. The inoculum, in addition to the oospores, consists of a thin walled mycelium under the hourglass cell layer of the seed coat and a thick walled resting mycelium in the oospore crust on the seed surface. By a modified Feulgen staining technique it was possible to demonstrate that the thin walled and the thick walled resting mycelium as well as thin and thick walled oogonia contain nuclei which are Feulgen positive suggesting that portions of the mycelium of P. manshurica may survive even on the surface of dry, mature seeds.     

The Influence of the Inoculum Source of Rhizoctonia solani on Development of Black Scurf on Potato

Rhizoctonia solani, the causal agent of stem canker and black scurf on potato, survives as sclerotia on tubers, in soil and in plant residues. The objective of the present study was to evaluate the importance of inoculum source on disease development. Disease‐free minitubers and seed tubers contaminated with low levels of R. solani were planted in fumigated or artificially inoculated growth mixture in greenhouse experiments. Black scurf incidence and severity were significantly higher when the inoculum was present in both seed tubers and soil, compared with either of them separately. The severity of disease symptoms on the subterranean parts of the plant also were significantly higher in plots where both seed tubers and soil were contaminated, compared with plots where the inoculum source was either the seed tubers or the soil. Thus, both major sources of inoculum, seed tubers and soil, are important in disease development. However, when both sources are present, black scurf incidence and severity are increased, leading to economical damage to tuber yield and quality. Additional results from field trials support these findings. Disease incidence and severity on daughter tubers were correlated with levels of contamination in seed tubers and soil. When seed tubers and soil were heavily infested, the levels of black scurf incidence and severity on daughter tubers were very high; when seed tuber and soil infestation were very low, black scurf incidence and severity on progeny were also lower. Disease levels were reduced by in‐furrow fungicide treatment, but were less effective when the initial levels of the fungus on the seed tubers and in the soil were high.     

Stem canker (Rhizoctonia solani) of maincrop potatoes.

In two years, potato plants were sampled at 1- or 2- weekly intervals from plots planted with seed tubers bearing sclerotia of Rhizoctonia solani (black scurf) and with seed without sclerotia either infested or not with cultures of R. solani at planting. Sprouted King Edward seed was used in 1981 and sprouted and non-sprouted King Edward and Pentland Crown seed in 1982. In both years 60–80% of shoots from seed with sclerotia and 90% of shoots from seed inoculated at planting were affected with stem canker. Most disease developed before shoots emerged although it gradually increased later when new shoots arising both from seed tubers or as branches on shoots with damaged apices (pruned shoots) became infected before they emerged. Sprouting seed tubers bearing sclerotia decreased the disease on both cultivars but with soil-applied inoculum the disease was more severe on plants from sprouted than non-sprouted seed. Some stolons were infected by R. solani soon after they developed and incidence of infection later increased. Thirty to 50% of stolons were infected on plants from infected seed tubers and 60% on plants with soil-applied inoculum. With both cultivars and sources of inoculum about 70% of the infected stolons had their apices killed (pruned).     

低温纤维素酶菌株CNY086发酵条件优化(Ⅱ)

本文是在前文(Ⅰ)确定菌株CNY086低温纤维素酶发酵培养基的基础上,通过单因素和正交实验研究温度、装液量、接种量及种龄对菌株CNY086低温纤维素酶发酵影响.最适发酵温度、装液量、接种量和种龄分别为15℃、250 mL/500 mL、15%、10 h.上述条件下CNY086菌株5 L发酵酶活力为104.36 U/mL.     

Transmission of Alternaria macrospora in Cotton Seeds

Alternaria macrospora was isolated from seeds only after the natural opening of the bolls and exposure of seeds to an environment in which the fungus was present. The fungus lacks the ability to penetrate the boll wall and reach the seed site. Attempts to isolate the pathogen from seeds of immature bolls at different developmental stages failed. Internal infection by slow injection resulted in seed infection and partial shedding of the injected plant parts which was high in buds and decreased with the ripening to mature bolls. Severity of seed infection was not dependent on either inoculum level, boll physiological age or even if the boll itself was not diseased. Infection of flowers under field conditions caused flower shedding. Naturally infected seeds or inoculated seeds with inoculum levels of 100 spores/ml and above resulted in diseased cotyledons, the incidence of which was, for inoculated seeds, positively correlated with inoculum level. A small difference was observed between cultivars in susceptibility to artificial inoculation at the cotyledon stage. A. macrospora survived on commercial cotton seeds and on post-season plants left growing at the field edges. Survival in plant debris under field conditions was minimal and may only have a minor effect on field reinfestation.     

Fungicidal control of Macrophomina phaseolina altered in pathogenicity by substrate nutrients

The presence of different carbon and nitrogen sources and bivalent metal compounds in the substrate medium influenced the mycelial growth of Macrophomina phaseolina and in vitro susceptibility to fungicides. The inoculum from such substrate media showed differences in pathogenicity on mung bean (Vigna radiata). Sucrose and asparagine significantly increased the mycelial growth as well as pathogenicity of the fungus. Absence of bivalent metal ions, viz., Fe⁺⁺, Zn⁺⁺ and Mg⁺⁺ in the medium produced inoculum which caused maximum seedling mortality and foliage blight. Carbendazim and thiophanate-M as seed treatments were significantly less effective when the inoculum was from a medium containing glucose than when the inoculum was from a medium containing sucrose. Captafol and thiram gave significantly better disease control on mung bean when the inoculum used for soil inoculations was from media containing asparagine and ammonium nitrate compared to the inoculum grown on a medium containing sodium nitrate. Carbendazim, thiophanate-M, PMA, captafol and thiram gave good disease control when the inoculum used was raised on a medium devoid of bivalent metal ions. Carbendazim and thiophanate-M were the best fungicides as foliar treatments and controlled the disease irrespective of carbon, nitrogen and bivalent metal ion status of the substrate medium used for the production of inoculum.     

The development of silver scurf (Helminthosporium solani) disease of potato

The main source of inoculum of Helminthosporium solani was the seed tuber; the fungus was not detected in soils in which infected potatoes had been grown the previous year. Lesions spread rapidly on seed tubers after planting, so that within a few weeks the entire surface was covered. As lesions aged they lost the capacity to sporulate, so most inoculum was produced in the first few weeks after planting. Infection of progeny tubers was first detected at the heel (stolon attachment) end soon after their initiation. Once established, lesions spread slowly on the surface of progeny tubers when in the soil; more rapid spread took place during storage. Potatoes which seemed unaffected at lifting became diseased during storage.     

Grass seed infection following inundation with Pyrenophora semeniperda

Seed infection by the seed-borne pathogen Pyrenophora semeniperda was demonstrated following inundative applications of the fungus to several annual grass weeds and wheat. The optimum time for applying inoculum was found to be around mid-anthesis in wheat. Applications of inoculum during and after anthesis in field experiments resulted in seed infection that manifested as stromatal development which affected germination or reduced seedling vigour. In an initial field experiment, >70% of Bromus diandrus seeds exhibited stromata of P. semeniperda. In a field comparison of inoculum types, conidial suspensions resulted in the greatest level of seed infection of B. diandrus compared with several types of inoculum containing mycelium fragments. Inundation of Avena fatua, Lolium rigidum, Hordeum leporinum, Vulpia bromoides and B. diandrus under field conditions with conidia of P. semeniperda also resulted in either the failure of infected seeds to germinate or a reduction of seedling vigour. It is concluded that the use of P. semeniperda as a seed-borne bioherbicide may be a biologically reasonable tactic, and the many logistical and technological constraints impeding its development are discussed.     

Celery leaf spot: sources of inoculum

The relative importance of infected celery seed, infected leaf debris in the soil, and infected wild celery, in the incidence of Septoria leaf spot in cultivated celery has been investigated. Infection can be caused when the sole source of inoculum is viable spores on the seed surface; such spores are considered to be the main cause of disease outbreaks. Of all the seed samples examined, 93% were infected by Septoria spp. In untreated seed samples, 40% carried viable spores which survived for up to 15 months on seed stored in the laboratory, and for longer periods on seed stored at -20d? C. However, ageing of seed is not recommended as a commercial control measure. The fungus was not found in seed embryos or endosperms but mycelium was present in pericarps and testas. Unconfirmed evidence suggests that in favourable circumstances new spores might be produced in old seed-borne pycnidia.     

Effects of Self-sown Wheat on Levels of the Take-all Disease on Seedlings of Winter Wheat Grown in a Model System

Abstract The mechanisms of build-up of inoculum of the take-all fungus, Gaeumannomyces graminis var. tritici. and infection of crop plants from self-sown (volunteer) wheat were analysed in a factorial experiment in a glasshouse. Treatments comprised sowing-date, inoculum density, soil aeration and texture, volunteer density and control of volunteers. Timing of treatments was related to field practice by the use of cumulative day-degrees and by consideration of sowing date and the geometry of seed placement. Prior sowing of volunteers, which were exposed to soil inoculum. resulted in significant increases in the incidence and severity of disease on subsequently sown seedlings. Increasing the density of volunteer seedlings increased the levels of subsequent infection. This effect, however, was significantly influenced by sowing date and density of the initial inoculum. The use of glyphosate to kill volunteers did not markedly affect the carry-over of disease.     

Influence of inoculum density of the antagonistic bacteria Pseudomonas fluorescens and Pseudomonas corrugata on sugar beet seedling colonisation and suppression of Pythium damping off

The effect of initial inoculum density of the antagonistic bacterial strains Pseudomonas fluorescens B5 and Pseudomonas corrugata 2140 (10³ to 10⁸ CFU per seed pellet) on sugar beet seedling colonisation, in situ bioluminescence and antagonistic activity towards Pythium ultimum was investigated. Populations of the bacteria colonising sugar beet root systems approached an apparent carrying capacity of 10⁵ to 10⁶ CFU per plant after 12 d growth, irrespective of inoculum density. This meant an up to 320-fold population increase at low inoculum densities and a decrease at high densities. Population densities of both bacteria and their corresponding in situ bioluminescence (resulting from luciferase enzyme expression from the inserted luxAB genes) reached highest levels in the hypocotyl region and in the upper root region 0–20 mm below seed level (10⁴–10⁶ CFU/cm section, 10¹–10³ RLU/cm section) and decreased with root depth. In situ bioluminescence, which indicates physiological activity, was measurable at lowest antagonist initial inoculum density (10³ CFU per seed pellet) and did not increase significantly with increasing inoculum density. Bioluminescence was also significantly correlated with population density. For Pseudomonas fluorescens B5, the total population size per plant and downward colonisation of the root (below 40 mm depth) increased significantly with antagonist inoculum density applied to the seeds. For Pseudomonas corrugata 2140, no significant influence of initial inoculum density on root colonisation was observable. Survival and dry weight of sugar beet seedlings in Pythium infested soil increased significantly with increasing inoculum density of Pseudomonas fluorescens B5, whereas for Pseudomonas corrugata 2140, initial densities of 10⁴ to 10⁶ CFU per seed resulted in maximal survival of plants.     

Solid-state fermentation for the production of Monascus pigments from jackfruit seed

The aim of the present work was to investigate the feasibility of jackfruit seed powder as a substrate for the production of pigments by Monascus purpureus in solid-state fermentation (SSF). A pigment yield of 25ODUnits/g dry fermented substrate was achieved by employing jackfruit seed powder with optimized process parameters such as 50% initial moisture content, incubation temperature 30 degrees C, 9x10(4)spores/g dry substrate inoculum and an incubation period of seven days. The color of the pigments was stable over a wide range of pH, apparently due to the buffering nature of the substrate, which could be a significant point for its scope in food applications. To the best of our knowledge this is the first report on pigment production using jackfruit seed powder in solid-state fermentation (SSF).     

Factors affecting host range in a generalist seed pathogen of semi-arid shrublands

Generalist pathogens can exhibit differential success on different hosts, resulting in complex host range patterns. Several factors operate to reduce realized host range relative to potential host range, particularly under field conditions. We explored factors influencing host range of the naturally occurring generalist ascomycete grass seed pathogen Pyrenophora semeniperda. We measured potential host range in laboratory experiments at high inoculum loads with 26 grass species, including the primary host Bromus tectorum, and developed models to predict susceptibility and tolerance based on host traits, including germination speed, seed hardness, seed size, and phylogenetic relations. We also examined pathogen and host density effects on infection and mortality. All species tested were at least somewhat susceptible to the pathogen at high inoculum loads, but both infection and mortality varied widely. Species more closely related to the original host (B. tectorum) were more susceptible to infection, whereas species with slower germination were less tolerant and therefore more likely to suffer mortality. Infection and mortality were sharply reduced as inoculum load was reduced. Intermediate loads had major negative impacts on dormant B. tectorum seeds but generally minimal effects on native species. In addition, field seed bank studies determined that P. semeniperda rarely exploits native grass species as hosts. This marked reduction in realized host range relative to potential host range indicates that laboratory host range studies are potentially a poor predictor of either the current or possible future realized host range for wildland plant pathogens.     

Epidemiological analysis of risk factors for the spread of potato viruses in Switzerland

Virus diseases represent important economic threats to the production of seed potatoes worldwide, yet little quantitative information is available on the relative merits of different measures of virus control applied singly or in combination. In this study, we compiled data from the national seed certification programme on the incidence of Potato Virus Y (PVY) and Potato Leaf‐roll Virus (PLRV) in potato tubers in Switzerland for the years 1990–2009 and used generalised linear models to investigate the influence of key epidemiological factors on infection risk. Results showed that post‐harvest virus incidence increased with initial inoculum levels, with the largest change in infection risk occurring between fields with no inoculum and those with low levels of inoculum. Virus incidence decreased with increasing altitude of fields. Surprisingly, infection risk was considerably lower for imported seed lots even though the model controlled for the effect of inoculum level, potato variety and other confounders. Overall, variety was the most important factor influencing virus risk. The results of the present analysis are useful to fine‐tune decision‐support systems that predict disease risk under different epidemiological scenarios.     

Slash Pile Burning Effects on Soil Biotic and Chemical Properties and Plant Establishment: Recommendations for Amelioration

Ponderosa pine forest restoration consists of thinning trees and reintroducing prescribed fire to reduce unnaturally high tree densities and fuel loads to restore ecosystem structure and function. A current issue in ponderosa pine restoration is what to do with the large quantity of slash that is created from thinning dense forest stands. Slash piling burning is currently the preferred method of slash removal because it allows land managers to burn large quantities of slash in a more controlled environment in comparison with broadcast burning slash. However burning slash piles is known to have adverse effects such as soil sterilization and exotic species establishment. This study investigated the effects of slash pile burning on soil biotic and chemical variables and early herbaceous succession on burned slash pile areas. Slash piles were created following tree thinning in two adjacent approximately 20‐ha ponderosa pine (Pinus ponderosa) restoration treatments in the Coconino National Forest near Flagstaff, Arizona. We selected 30 burned slash pile areas and sampled across a gradient of the burned piles for arbuscular mycorrhizal (AM) propagule densities, the soil seed bank, and soil chemical properties. In addition, we established five 1‐m² plots in each burned pile to quantify the effect of living soil (AM inoculum) and seeding amendments on early herbaceous succession in burned slash pile areas. The five treatments consisted of a control (no treatment), living soil (AM inoculum) amendment, sterilized soil (no AM inoculum) amendment, seed amendment, and a seed/soil (AM inoculum) amendment. Slash pile burning nearly eliminated populations of viable seeds and AM propagules and altered soil chemical properties. Amending scars with native seeds increased the cover of native forbs and grasses. Furthermore adding both seed and living soil more than doubled total native plant cover and decreased ruderal and exotic plant cover. These results indicate that seed/soil amendments that increase native forbs and grasses may enhance the rate of succession in burned slash pile areas by allowing these species to outcompete exotic and ruderal species also establishing at the site through natural regeneration.