Production of the fungal biocontrol agent Epicoccum nigrum by solid substrate fermentation: effect of water activity on accumulation of compatible solutes

Epicoccum nigrum conidia were produced by solid fermentation on wheat grains (cv. Rendeveaux and Brigadier) at different water activities (a_w). Conidial production was highest at high a_w(0.996) than at reduced a_w (0.98). However, conidial production at reduced a_w was improved when the a_w of the substrate was adjusted with a mixture of glycerol and water. Maximum levels ofconidiation were 7–11 × 10⁶ conidia g⁻¹ grain. The a_w of the solid substrate affected the pattern of accumulation of compatible solutes in the conidia. Mannitol was the main polyol in all conidialtypes. However, the amounts of mannitol were higher in conidia produced at high a_w. At reduced a_w the conidia of E. nigrum accumulated moreglycerol, which is more efficient in the osmorregulation proccess than mannitol. Arabitol accumulated in low amounts, specifically in conidia produced at the lower a_w, on cv. Rendeveaux but not on cv. Brigadier. Trehalose was detected in higher amounts in cv. Rendeveaux than in cv. Brigadier, andthe amounts were higher in conidia produced at high a_w. A significant amount of endogenous solutes was detected in the washing liquid used for the separation of the conidia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Osmotic stress adaptation, compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat

Fusarium head blight (FHB) caused by Gibberella zeae (anamorph = Fusarium graminearum) is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. Biological control has been demonstrated to be effective under laboratory conditions but a few biocontrol products have been effective under field conditions. The improvement in the physiological quality of biocontrol agents may improve survival under field conditions, and therefore, enhance biocontrol activity. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were isolated from wheat anthers and showed significant effect on control of FHB under greenhouse assays. This study showed the effect of water availability measured as water activity (a_W) using a growth medium modified with NaCl, glycerol and glucose on: (i) osmotic stress tolerance, (ii) viability in modified liquid medium, (iii) quantitative intracellular accumulation of betaine and ectoine and (iv) the biocontrol efficacy of the physiologically improved agents. Viability of B. subtilis RC 218 in NaCl modified media was similar to the control. Brevibacillus sp. RC 263 showed a limited adaptation to growth in osmotic stress. Betaine was detected in high levels in modified cells but ectoine accumulation was similar to the control cells. Biocontrol activity was studied in greenhouse assays on wheat inoculated at anthesis period with F. graminearum RC 276. Treatments with modified bacteria reduced disease severity from 60% for the control to below 20%. The physiological improvement of biocontrol agents could be an effective strategy to enhance stress tolerance and biocontrol activity under fluctuating environmental conditions.     

Osmoregulation, growth and sucrose accumulation in germinated Trigonella foenum-graecum (fenugreek) seeds treated with polyethylene glycol

Germinated seeds of Trigonella foenum-graecum L. (fenugreek) were grown in water or in polyethylene glycol (PEG) solutions. After endosperm removal, the water relations, growth, dry weight, sucrose and reducing sugar content of the embryo were determined. Under water sstress conditions, water content and osmotic potential (π₀) at saturation, growth and dry weight were lower than in non-stressed controls. The reduction in dry weight indicated a lower uptake of solutes from the endosperm and the decrease in π₀ was not accompanied by an increase in the amount of the accumulated solutes. It is suggested that embryos of stressed fenugreek seeds control osmotic potential by reduction of water uptake and that this results in reduction of growth. Embryos isolated from germinated seeds ("naked" embryos) were grown in water or in PEG solutions, with or without galactose (as an external solute source substituting for the endosperm). The results indicate that a decrease in the external solute did not account for growth reduction under conditions of water stress, and that decreased solute transport to the embryo may be important. The sucrose contents of "naked" embryos and of embryos from whole seeds were higher after PEG treatment, while reducing sugar contents were lower compared to non-stressed controls. The increased sucrose accumulation may be due to decreased sucrose hydrolysis.     

Osmotic and matric potential effects on growth, sugar alcohol and sugar accumulation by Aspergillus section Flavi strains from Argentina

AIMS: The effect of osmotic and matric potential stress on growth and sugar alcohols (polyols: glycerol, erythritol, arabitol and mannitol) and sugars (trehalose and glucose) accumulation in toxigenic and nontoxigenic colonies of Aspergillus flavus and A. parasiticus was evaluated. METHODS AND RESULTS: Growth of Aspergillus section Flavi with significant reductions at 20 and 30 degrees C was more sensitive to changes in matric potential, between 60 and 100% in the range of -7 to -14 MPa. No significant differences were found between toxigenic and nontoxigenic strains for both species. Total polyol accumulation in unamended maize meal agar medium (-0.75 MPa water potential) was higher at 30 than 20 degrees C. The major change in concentrations of endogenous sugars and total polyols was in matrically amended medium (with PEG 8000) at -7 and -10 MPa. Accumulation of glucose, arabitol, mannitol and erythritol content of A. flavus and A. parasiticus mycelial colonies was greater in normal unstressed maize meal agar medium (-0.75 Mpa) at 20 degrees C. This was modified by solute and matric stress. CONCLUSIONS: The data showed relative sensitivity to osmotic and matric potential, and temperature, and the impact on growth rates, polyol and sugar accumulation in mycelia of A. flavus and A. parasiticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The matric potential effects on growth may be of particular importance for growth and survival in environments with low-matric potential stress. The tolerance of spoilage fungi such as Aspergillus section Flavi to such modifications could increase the potential for spoilage and mycotoxin production in such substrates. This knowledge is important for understanding the relative ecological fitness of these aflatoxigenic species and in the development of prevention strategies for their control.     

Accumulation of the compatible solutes, glycine-betaine and ectoine, in osmotic stress adaptation and heat shock cross-protection in the biocontrol agent Pantoea agglomerans CPA-2

AIMS: The effect of modifying the water activity (a(w)) of Pantoea agglomerans growth medium with the ionic solute NaCl on water stress resistance, heat-shock survival and intracellular accumulation of the compatible solutes glycine-betaine and ectoine were determined. METHODS AND RESULTS: The bacterium was cultured in an unmodified liquid medium or that modified with NaCl to 0.98 and 0.97 a(w), and viability of cells evaluated on a 0.96 a(w)-modified solid media to check water stress tolerance. Cells grown under ionic stress had better water stress tolerance than control cells. These cells also had cross-protection to heat stress (30 min, 45 degrees C). The modified cells accumulated substantial amounts of the compatible solutes glycine-betaine and ectoine in contrast to the control cells, which contained little or none of these two compounds. CONCLUSIONS: Improvement in osmotic and thermal tolerance of cells of the biocontrol agent P. agglomerans by modifying growth media with the ionic solute NaCl was achieved. The compatible solutes glycine-betaine and ectoine play a critical role in environmental stress tolerance improvement. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach provides a method for improving the physiological quality of inocula and could have implications for formulation and shelf-life of biocontrol agents.     

重金属复合胁迫下碱蓬萌发生长及富集特征

为探究重金属复合胁迫对金川镍铜矿区广布植物碱蓬Suaeda salsa(L.)Pall.的影响,根据当地环境条件及预实验结果设置胁迫梯度,测定分析重金属胁迫下碱蓬种子萌发和芽期生理指标,并从野外站台取样研究碱蓬重金属富集能力。结果表明:无论是单一胁迫还是低浓度复合胁迫(Cu20和Ni20复合),发芽期碱蓬的生长均呈现"低促高抑"的趋势,即低浓度(≤40 mg/L)时促进碱蓬生长,高浓度(≥80 mg/L)时抑制碱蓬生长;高浓度复合胁迫(Cu320和Ni320复合)下均抑制碱蓬的生长。MDA(丙二醛)的含量随胁迫浓度的增加而增加;胁迫组可溶性蛋白和游离脯氨酸含量整体上高于对照组;单一胁迫下POD(过氧化物酶)活性随胁迫浓度增加而增加;复合胁迫下低浓度提高POD活性,高浓度抑制POD活性;碱蓬叶片的平均转移系数(TF)大于茎部,且平均转移系数大于1.00;碱蓬叶片的富集系数(BCF)大于根部大于茎部。碱蓬对Cu和Ni均具有很高的耐受性,但对于Cu的耐性强于Ni;低浓度时Cu、Ni复合胁迫对碱蓬生长的促进作用强于Cu、Ni单一胁迫,高浓度时则相反;碱蓬具有较高的重金属富集和转移能力;在当前矿区土壤环境背景下,碱蓬可以作为矿区生态恢复和重金属污染修复的备选植物。     

Water potential and ionic effects on germination and seedling growth of two cold desert shrubs

We tested expectations that two desert shrubs would differ in germination and seedling relative growth rate (RGR) responses to Na and Ψ_s stress. The study species, Chrysothamnus nauseosus ssp. consimilis and Sarcobatus vermiculatus (hereafter referred to by genus), differ in their distribution along salinity gradients, with Chrysothamnus inhabiting only less saline areas. In growth chamber studies, declining Ψ_s (−0.82 to −2.71 MPa) inhibited germination of both species, and Chrysothamnus was less tolerant of Ψ_s stress than Sarcobatus. Germination fell below 10% for Chrysothamnus at −1.64 MPa (NaCl and PEG), and for Sarcobatus at −2.4 MPa PEG. Neither species exhibited ion toxicity. There was substantial ion enhancement for Sarcobatus in lower Ψ_s, allowing for 40% germination in −2.71 MPa NaCl. For seedling RGR, species were not different at −0.29 or −0.82 MPa (0 and 100 mmol/L NaCl, respectively), but Chrysothamnus RGR declined substantially at −1.3 MPa (200 mmol/L NaCl). The greater stress tolerance of Sarcobatus was not associated with a lower RGR under nonsaline conditions. Species differences in seed and seedling Ψ_s stress tolerance probably contribute to the restricted distribution of Chrysothamnus to less saline areas. The Na uptake of Sarcobatus seedlings enhances its ability to deal with declining Ψ_s and establish in more saline areas.     

Differential effects of interspecific interactions and water availability on survival, growth and fecundity of three congeneric grassland herbs

Fitness of individual plants and of populations depends on the rates of survival, growth and fecundity. This study tested whether vital rates were differentially affected by biotic interactions and water availability. The effects of manipulations of above-ground competition (through clipping) and water availability (through water addition) on the vital rates of seedlings of three species (Viola elatior Fries, Viola pumila Chaix and Viola stagnina Kit.) were analysed in dry, mesic and wet grasslands. Water addition and grassland type had the largest effects on survival (accounting for 41 and 24% of total variation, respectively) across species. Height growth rate was positively affected by grassland type (19%) and water addition (12%) and varied among species (8%), while leaf accumulation rates and reproduction were affected by grassland type and clipping. The data suggested facilitative effects of the canopy on seedling survival in the dry grassland. This study presents evidence that environmental conditions and biotic interactions may have differential effects on seedling survival, growth and reproduction. The findings highlight the complex interplay between spatial and temporal environmental variation and biotic interactions in structuring plant communities.     

Shelter availability affects behaviour, size-dependent and mean growth of juvenile Atlantic salmon

1. Anthropogenic disturbances of the physical habitat and corresponding effects on fish performance are key issues in stream conservation and restoration. Reduced habitat complexity because of increased sediment loadings and canalization is of particular importance, but it is not clear to what extent fish populations are influenced directly by changes in the physical environment, or indirectly through changes in the biotic environment affecting the food availability. 2. Here, we test for the direct effect of habitat complexity on the performance (growth) of juvenile Atlantic salmon by manipulating shelter availability (interstitial spaces in the substrate) across 20 semi‐natural stream channels without altering the substrate composition, and stocking each channel with a common density of fish. A simple method for measuring salmonid shelters using flexible PVC tubes was developed and tested. Daytime sheltering behaviour and growth rates were compared across the channels differing in shelter availability. 3. Measured shelter availability was strongly negatively correlated with observed number of fish not finding shelters and mass loss rates of the fish (growth performance) increased with decreasing number of measured shelters. Number and mean depth of interstitial spaces explained up to 68% and 24% of the among‐channel variation in sheltering behaviour and growth performance, respectively. Furthermore, negative effects of shelter reduction increased with fish body size. Thus, changes in habitat structure may even influence the size selection gradients. 4. Shelter availability is an easily measured variable, possibly affecting the population demographics and long‐term evolutionary processes, and is therefore a key habitat factor to be considered in stream restoration and habitat classification.     

Rapid and tissue-specific accumulation of solutes in the growth zone of barley leaves in response to salinity

The aim of the present study was to test whether rapid accumulation of solutes in response to salinity in leaf tissues of barley (Hordeum vulgare L.) contributes to recovery and maintenance of residual elongation growth. Addition of 100 mM NaCl to the root medium caused an immediate reduction close to zero in elongation velocity of the growing leaf 3. After 20–30 min, elongation velocity recovered suddenly, to 40–50% of the pre-stress level. Bulk osmolality increased first, after 60 min, significantly in the proximal half of the elongation zone. Over the following 3 days, osmolality increases became significant in the distal half of the elongation zone, the adjacent, enclosed non-elongation zone and finally in the emerged portion of the blade. The developmental gradient and time course in osmolality increase along the growing leaf was reflected in the pattern of solute (Cl, Na and K) accumulation in bulk tissue and epidermal cells. The partitioning of newly accumulated solutes between epidermis and bulk tissue changed with time. Even though solute accumulation does not contribute to the sudden and partial growth recovery 20–30 min after exposure to salt, it does facilitate residual growth from 1 h onwards. This is due to a high sink strength for solutes of the proximal part of the growth zone and its ability to accumulate solutes rapidly and at high rates.Abbreviations EDX analysis Energy-dispersive X-ray analysis - LEV Leaf elongation velocity - LVDT Linear variable differential transformer - REGR Relative elemental growth rate     

Water availability and population origin affect the expression of the tradeoff between reproduction and growth in Plantago coronopus

Investment in reproduction and growth represent a classic tradeoff with implication for life history evolution. The local environment can play a major role in the magnitude and evolutionary consequences of such a tradeoff. Here, we examined the investment in reproductive and vegetative tissue in 40 maternal half‐sib families from four different populations of the herb Plantago coronopus growing in either a dry or wet greenhouse environment. Plants originated from populations with an annual or a perennial life form, with annuals prevailing in drier habitats with greater seasonal variation in both temperature and precipitation. We found that water availability affected the expression of the tradeoff (both phenotypic and genetic) between reproduction and growth, being most accentuated under dry condition. However, populations responded very differently to water treatments. Plants from annual populations showed a similar response to drought condition with little variation among maternal families, suggesting a history of selection favouring genotypes with high allocation to reproduction when water availability is low. Plants from annual populations also expressed the highest level of plasticity. For the perennial populations, one showed a large variation among maternal families in resource allocation and expressed significant negative genetic correlations between reproductive and vegetative biomass under drought. The other perennial population showed less variation in response to treatment and had trait values similar to those of the annuals, although it was significantly less plastic. We stress the importance of considering intraspecific variation in response to environmental change such as drought, as conspecific plants exhibited very different abilities and strategies to respond to high versus low water availability even among geographically close populations.     

Water deficit enhancement of proline and α-amino nitrogen accumulation in potato plants and its association with susceptibility to drought

Potato plants ( Solanum tuberosum L. cvs 'Up-to-Date', 'Desiree', 'Alpha', 'Spunta', 'Elvira' and 'Troubadour') were exposed to cycles of water stress and relief during growth. Severe water deficit induced increased proline content 6- to 7-fold in nonturgid leaves which just started to wilt, and 8- to 27-fold in fully wilted leaves of potatoes. However, proline content was not affected during the early stages of stress development over a range of osmotic potentials in the leaves. The rising proline content was related to turgor loss of leaves independent of changes in the osmotic potentials, which indicates that proline involvement in osmoregulation of potato leaves is unlikely.
Repeated cycles of water stress and relief resulted in increased proline and α-amino nitrogen content in the tuber tissue of some of the cultivars. The smallest increase in proline content was obtained in 'Alpha' tubers and the content of α-amino nitrogen remained unaffected by the water stress. Concomitantly, 'Alpha' was the most drought-tolerant cultivar, as determined by its capacity to accumulate dry matter in tubers under stress conditions. On the other hand, in tubers of cultivars which were more susceptible to drought, a marked increase in proline and α-amino nitrogen was observed in response to water stress. The possible association of these findings with tolerance of potatoes to repeated short periods of drought is discussed.     

Interactive effects of salinity and phosphorus availability on growth, water relations, nutritional status and photosynthetic activity of barley (Hordeum vulgare L.)

The interactive effects of salinity and phosphorus availability on growth, water relations, nutritional status and photosynthetic activity were investigated in barley (Hordeum vulgare L. cv. Manel). Seedlings were grown hydroponically under low or sufficient phosphorus (P) supply (5 or 180 μmol KH(2) PO(4) plant(-1) week(-1) , respectively), with or without 100 mm NaCl. Phosphorus deficiency or salinity significantly decreased whole plant growth, leaf water content, leaf osmotic potential and gas exchange parameters, with a more marked impact of P stress. The effect of both stresses was not additive since the response of plants to combined salinity and P deficiency was similar to that of plants grown under P deficiency alone. In addition, salt-treated plants exposed to P deficiency showed higher salt tolerance compared to plants grown with sufficient P supply. This was related to plant ability to significantly increase root:shoot DW ratio, root length, K(+)/Na(+) ratio, leaf proline and soluble sugar concentrations and total non-enzymatic antioxidant capacity, together with restricting Na(+) accumulation in the upper leaves. As a whole, our results indicate that under concomitant exposure to both salt and P deficiency, the impact of the latter constraint is pre-dominant.     

Growth, Water Content, and Proline Accumulation in Drought-Stressed Callus of Date Palm

This study was conducted to examine the response of date palm (Phoenix dactylifera L., cvs. Barhee and Hillali) calli to water stress. Callus derived from shoot tip explants was inoculated in liquid Murashige and Skoog medium containing 10 mg dm^–3-naphthaleneacetic acid, 1.5 mg dm^–3 2-isopentenyladenine, and 0 to 30 % (m/v) polyethylene glycol (PEG 8000) to examine the effect of water stress. After 2 weeks, callus growth, water content, and proline accumulation were assessed. Increasing water stress caused a progressive reduction in growth as expressed in callus fresh mass, relative growth rate, and index of tolerance. Both genotypes tested followed this general trend, however, cv. Barhee was more tolerant to drought stress than cv. Hillali. Increasing PEG concentration was also associated with a progressive reduction in water content and increased content of endogenous free proline.     

Water activity,temperature, and pH effects on growth of the biocontrol agent Pantoea agglomerans CPA-2

The growth response of the biocontrol agent Pantoea agglomerans to changes in water activity (a(w)), temperature, and pH was determined in vitro in nutrient yeast extract-sucrose medium. The minimum temperature at which P. agglomerans was able to grow was 267-272 kelvins (-6 to -1 degrees C), and growth of P. agglomerans did not change at varying pH levels (4.5-8.6). The minimum a(w) for growth was 0.96 in media modified with glycerol and 0.95 in media modified with NaCl or glucose. Solute used to reduce water activity had a great influence on bacterial growth, especially at unfavourable conditions (e.g., low pH or temperature). NaCl stimulated bacterial growth under optimum temperatures but inhibited it under unfavourable pH conditions (4.5 or 8.6). In contrast, the presence of glucose in the medium allowed P. agglomerans to grow over a broad range of temperature (3-42 degrees C) or pH (5-8.6) regimes. This study has defined the range of environmental conditions (a(w), pH, and temperature) over which the bacteria may be developed for biological control of postharvest diseases.     

Phylloplane microorganisms as a potential biocontrol agent against Helminthosporium oryzae Breda de Hann,the incitant of rice brown spot

Abstract

Fifteen pathogenic isolates of Helminthosporium oryzae were established from 15 different rice growing areas representing nine districts: Madurai, Theni, Thanjavur, Thiruvarur, Nagapatinum, Tirunelveli, Trichy, Ariyalur and Cuddalore district of Tamil Nadu. The symptoms initially appeared on the leaves as small, oval, dark brown to black spots. Under favourable conditions, the fungus attacks grain and caused grain discoloration. The isolate, collected from Ammapettai of Thanjavur district was the most virulent (Grade 7.47, PDI 82.96) on rice plants followed by Trichy isolate I₂ (Grade 7.26; PDI 80.74) while I₁₅ (Cumbum) was the least virulent (Grade 1.8, PDI 20.00). The isolates of H. oryzae varied in size (length and width) of the conidia, colour and the number of cells per conidium. The maximum (39.1 μm) length of the conidium was observed in I₁₀ followed by I₁₁ (37.3 μm). The maximum (17.1 μm) conidial width was observed in I₈. The colour of the conidium varied from light brown (isolates I₃, I₆ and I₁₄) to brown, while there was no appreciable difference in the conidial shape. The number of cells varied from 2 – 4 among the isolates. Six phylloplane microorganisms were tested against the virulent isolate of H. oryzae. Among them, Cladasporium spp was very effective in inhibiting the mycelial growth and spore germination of H. oryzae followed by Penicillum spp and Aspergillus flavus while Bacillus subtilis was least effective in inhibiting the mycelial growth and spore germination of H. oryzae.     

悬浮培养红豆杉细胞活力及存活率与生长周期的关系

用TTC法制定红豆杉细胞活力,用中性红测细胞存活率,探讨生长周期中细胞活力及存活率的变化规律。结果显示延迟期细胞活力最强,对数生长期活力较低,稳定期活力最低;而细胞存活率在整个周期中基本保持在70%-80%。     

Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis

In this study we propose revised structures for the two major compatible solutes of Rhodothermus marinus. We have also examined the accumulation of compatible solutes by the type strains of the slightly halophilic and thermophilic species Rhodothermus marinus and Rhodothermus obamensis at several growth temperatures and salinities. The major solutes of R. marinus were identified as α-mannosylglycerate (α-MG) and α-mannosylglyceramide (α-MGA), whereas R. obamensis accumulated only α-mannosylglycerate. The total osmolyte content was higher during the early exponential phase and decreased abruptly as growth continued into the stationary phase. At low growth temperatures, R. marinus responded to water stress by accumulation of α-mannosylglycerate and its amide, in addition to low levels of trehalose, glutamate, and glucose. At the highest growth temperature, α-mannosylglycerate was the major compatible solute and α-mannosylglyceramide was not detected. When both compounds were present, an increase in the salinity of the growth medium favored the accumulation of α-mannosylglyceramide over α-mannosylglycerate. The absence of α-mannosylglyceramide in R. obamensis at all growth temperatures and salinities constituted the most pronounced difference in the profiles of compatible solute accumulation by the two strains. Trehalose was also a prominent solute in this organism. Both organisms accumulated higher levels of α-mannosylglycerate as the temperature was raised. The importance of the two compounds in the mechanisms of thermoadaptation and osmoadaptation is discussed. Received: February 10, 1998 / Accepted: January 11, 1999     

Restoring the Victorian Western (Basalt) Plains grassland. 1. Laboratory trials of viability and germination, and the implications for direct seeding

Summary   The Victorian Western (Basalt) Plains grassland is one of Australia's most threatened plant communities. Practitioners using seed for its restoration need to know whether seed can be sown fresh or whether it requires an after-ripening period. This study assessed the viability and germination of freshly harvested wild seed from 64 grassland species indigenous to the Basalt Plains of western Victoria. The seed was collected as part of a broader experiment that examined the potential of direct-sown complex seed mixes for the restoration of grassland communities. The germination of fresh seed at 25°C varied widely between species. Comparisons with tetrazolium viability tests for each species indicated varying levels of dormancy within the species pool. Germination separated into three broad responses at day 28. One-third of the species failed to germinate, one-third germinated at 1% to 50% and the remaining species germinated between 51% and 100%. Therefore, if the aim of a sowing was the rapid and synchronous establishment of most of the sown species, the use of fresh seed in restoration could be problematic. After 3 months of dry storage, eight species were re-tested for germination. Each of the selected species had shown high viability but low initial germination. Only two species significantly increased their total germination at 25°C. The annual species, Triptilodiscus pygmaeus , increased its total germination from 6% as fresh seed to 99% after dry storage. Testing the viability and germination capacity of freshly harvested seed from a large and diverse sample of native grassland species demonstrated that many of the species were unlikely to germinate rapidly or synchronously when sown in complex seed mixes soon after harvest. This finding has implications for the scheduling and management of restoration projects that rely on the use of such seed.