The effect of volatile and gaseous metabolites of swelling seeds on germination of fungal spores

Effects of volatile and gaseous metabolites of swelling seeds of pea, bean, wheat, corn, cucumber, tomato, lentil, carrot, red pepper and lettuce on germination of spores of five genera of fungi were found to depend rather on the fungal than on the plant genus. Germination of spores ofBotrytis cinerea, Mucor racemosus andTrichoderma viride was most severely inhibited. Spores ofVerticillium dahliae were less sensitive and germination of spores ofFusarium oxysporum was inhibited only in two cases. On the other hand, exudates of pea and bean stimulated germination of spores ofFusarium oxysporum. Also spores ofTrichoderma viride germinated better in an atmosphere enriched with exuded metabolites of swelling lettuce seeds. When carbon dioxide produced by the swelling seeds was absorbed in potassium hydroxide, spores ofTrichoderma viride andVerticillium dahliae did not germinate at all, the inhibitory effects of volatile and gaseous exudates on germination of spores ofMucor racemosus were accentuated, and also the percentage of germinated spores ofFusarium oxysporum decreased. Germination of spores ofBotrytis cinerea was not influenced. Absorption of volatile and gaseous metabolites in a solution of potassium permanganate decreased in most cases their inhibitory effects, particularly inBotrytis cinerea.     

Colonization by Arbuscular Mycorrhizal Fungi of Sorghum Leads to Reduced Germination and Subsequent Attachment and Emergence of Striga hermonthica

Two sorghum cultivars: the Striga-tolerant S-35 and the Striga-sensitive CK60-B were grown with or without arbuscular mycorrhizal (AM) fungi, and with or without phosphorus addition. At 24 and 45 days after sowing (DAS) of sorghum, root exudates were collected and tested for effects on germination of preconditioned Striga hermonthica seeds. Root exudates from AM sorghum plants induced lower germination of S. hermonthica seeds than exudates from non-mycorrhizal sorghum. The magnitude of this effect depended on the cultivar and harvest time. A significantly (88–97%) lower germination of S. hermonthica seeds upon exposure to root exudates from AM S-35 plants was observed at both harvest times whereas for AM inoculated CK60-B plants a significantly (41%) lower germination was observed only at 45 DAS. The number of S. hermonthica seedlings attached to and emerged on both sorghum cultivars were also lower in mycorrhizal than in non-mycorrhizal plants. Again, this reduction was more pronounced with S-35 than with CK60-B plants. There was no effect of phosphorus addition on Striga seed germination, attachment or emergence. We hypothesize that the negative effect of mycorrhizal colonization on Striga germination and on subsequent attachment and emergence is mediated through the production of signaling molecules (strigolactones) for AM fungi and parasitic plants.Key Words: arbuscular mycorrhiza, root exudate, sorghum, striga, strigolactones, germination     

Effect of volatile and gaseous metabolites of germinating pea seeds on micromycetes

Differences in the effect of volatile and gaseous metabolites of germinating pea seeds on the germination of spores of Mucor racemosus and macroconidia of Fusarium oxysporum are described. Germination of spores of M. racemosus was inhibited by seed metabolites whereas germination of macroconidia of F. oxysporum was stimulated during the first two days and inhibition occurred only after further two days of germination of the seeds. A pronounced inhibition of germination of spores of both micromycetes took place due to absorption of CO2 from volatile and gaseous metabolites. Absorption of some components of seed metabolites in a KMnO4 solution led to a decrease of the inhibitory effect on germination of spores of M. racemosus and stimulatory effect on germination of macroconidia of F. oxysporum.     

Sensitivities of Germinating Spores and Carvacrol-Adapted Vegetative Cells and Spores of Bacillus cereus to Nisin and Pulsed-Electric-Field Treatment

Treatment of Bacillus cereus spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage. In contrast, germinating spores were found to be sensitive to PEF treatment. Nisin treatment was more efficient than PEF treatment for inactivating germinating spores. PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated. Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment. Resistance to nisin was lost immediately when the germination process started. A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin. On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol. Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.     

Root exudates from lentil (Lens culinaris medic) seedlings in relation to wilt disease

Summary Root exudates from healthy and diseased lentil plants (Lens culinaris) have been investigated in relation to the wilt disease caused byFusarium oxysporum f.lentis. In all ten amino acids and five sugars have been detected. The spore germination of the pathogen in root exudates indicated that 21-days root exudate was inhibitory. Glycine and phenylalanine were detected in 21-days exudate and were found to have an inhibitory effect upon the germination of the spores of the pathogen which may partly explain the lesser disease incidence when plants of more than 3 weeks are inoculated with the pathogen.     

Secondary leaf fall of Hevea brasiliensis: factors affecting the production, germination and viability of spores of Colletotrichum gloeosporioides

The optimum temperature for growth and sporulation of Colletotrichum gloeosporioides from Hevea brasiliensis was between 26 and 32 ^oC, whereas spore germination exceeded 90% between 21.5 and 30.5 ^oC. Germination decreased in culture after 3 days, and on exposure of spores to sunlight or oven heat (46 ^oC) for 10 min. Spore viability and germination were sensitive to atmospheric humidity; at 99% r.h. germination was half that at 100% r.h. and was negligible below 97% r.h. Germination decreased by up to 30% after 3 h storage at 80% r.h. Continuous light favoured spore production in vitro, but spores produced in the dark had a higher percentage germination. No differences were detected between the numbers of spores germinating on leaves of different ages, although there were slightly more on susceptible cultivars and in the presence of extracts of uninfected susceptible leaves. Extracts from, infected leaves depressed spore germination, as did concentrations above 5 times 10⁵ spores/ml. The highest % germination was observed when naturally infected leaves were dry-stored for up to 20 days and then incubated for 2 days in a moist chamber.     

Mechanisms associated with tolerance to flooding during germination and early seedling growth in rice (Oryza sativa)

Background and Aims

Flooding slows seed germination, imposes fatalities and delays seedling establishment in direct-seeded rice. This study describes responses of contrasting rice genotypes subjected to flooding or low oxygen stress during germination and discusses the basis of tolerance shown by certain cultivars.

Methods

In one set of experiments, dry seeds were sown in soil and either watered normally or flooded with 10 cm of water. Seedling survival and shoot and root growth were assessed and seed portions of germinating seedlings were assayed for soluble sugars and starch concentrations. The whole germinating seedlings were assayed for amylase and peroxidase activities and for ethylene production. Activities of enzymes associated with anaerobic respiration were examined and gene expression was analysed separately with seeds germinating under different amounts of dissolved oxygen in dilute agar.

Key Results

Flooding during germination reduced survival but to a lesser extent in tolerant genotypes. Starch concentration in germinating seeds decreased while sugar concentration increased under flooding, but more so in tolerant genotypes. Amylase activity correlated positively with elongation (r = 0·85 for shoot and 0·83 for root length) and with plant survival (r = 0·92). Tolerant genotypes had higher amylase activity and higher RAmy3D gene expression. Ethylene was not detected in seeds within 2 d after sowing, but increased thereafter, with a greater increase in tolerant genotypes starting 3 d after sowing. Peroxidase activity was higher in germinating seeds of sensitive genotypes and correlated negatively with survival.

Conclusions

Under low oxygen stress, tolerant genotypes germinate, grow faster and more seedlings survive. They maintain their ability to use stored starch reserves through higher amylase activity and anaerobic respiration, have higher rates of ethylene production and lower peroxidase activity as germinating seeds and as seedlings. Relevance of these traits to tolerance of flooding during germination and early growth is discussed.Key words: Amylase, anoxia, crop establishment, direct-seeded rice, ethylene, flooding, germination, hypoxia, Oryza sativa     

Conditioning duration and agents involved in broomrape seeds responding to germination stimulants

Weedy broomrape species, such as sunflower broomrape (Orobanche cumana Wallr.) and Egyptian broomrape [Phelipanche aegyptiaca Pers. (syn. O. aegyptiaca)], require a period of pre-conditioning before they can respond to germination stimulants. Thus, the sensitivity of weedy broomrape seeds to germination stimulants could be an important factor for broomrape control. In this study, the influence of conditioning agents, conditioning period (0–21 days) and germination stimulants on the germination of sunflower broomrape and Egyptian broomrape seeds was analyzed. Without conditioning, the sunflower and Egyptian broomrape seeds exhibited negligible germination responses to the stimulants. The germination rate of the broomrape seeds increased rapidly with conditioning period and reached a maximum under a conditioning period of 4–10 days; further prolonged conditioning resulted in a decrease in the germination rate. Gibberellic acid (GA₃) could not only break the dormancy of the sunflower and Egyptian broomrape seeds but also maintained the high sensitivity of these seeds even after 21 days of conditioning. Furthermore, 100 µM of GA₃ induced the germination of the Egyptian broomrape seeds. The stimulants that induced Egyptian broomrape germination were ranked in decreasing order as GR24 (76.8?%), strigol (76.1?%), tobacco root exudates (49.5?%), dehydrocostus lactones (DCL, 39.2?%), and maize root exudates (18?%). In contrast, GA₃ did not directly induce sunflower broomrape seed germination, which responded to strigol (62.8?%)?>?maize root exudates (58.2?%)?>?GR24 (57.9?%)?>?tobacco root exudates (41.6?%)?>?DCL (41.3?%). These results indicate specialized recognition of germination stimulants by sunflower and Egyptian broomrape. This study may contribute to a better understanding of parasitic weed germination and may lead to improved control strategies.     

Ethylene antagonizes the inhibition of germination in Arabidopsis induced by salinity by modulating the concentration of hydrogen peroxide

The capacity of plants to achieve successful germination and early seedling establishment under high salinity is crucial for tolerance of plants to salt. The gaseous hormone ethylene has been implicated in modulating salt tolerance, but the detailed role of how ethylene modulates the response of early seedling establishment to salt is unclear. To better understand the role of the ethylene signal transduction pathway during germination and seedling establishment, an ethylene insensitive mutation (ein2-5) and an ethylene sensitive mutation (ctr1-1) of Arabidopsis were analyzed under saline conditions and compared with the wild type plant (Col-0) as control. High salinity (>100?mM NaCl) inhibited and delayed germination. These effects were more severe in the ethylene insensitive mutants (ein2-5) and less severe in the constitutive ethylene sensitive plants (ctr1-1) compared with Col-0 plants. Addition of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or inhibitors of ethylene action implied that ethylene was essential for early seedling establishment under normal and saline conditions. Salt stress increased the endogenous concentration of hydrogen peroxide (H₂O₂) in germinating seeds and ACC reduced its concentration. Our results suggest that ethylene promotes germination under salinity by modulating the endogenous concentration of H₂O₂ in germinating seeds. These findings demonstrate that ethylene is involved in regulating germination as an initiator of the process rather than consequence, and that ethylene promotes germination by modulating the endogenous concentration of H₂O₂ in germinating seeds under salinity.     

Biochemistry of fern spore germination: protease activity in ostrich fern spores

Protease activities were detected in quiescent and germinating spores of the ostrich fern (Matteuccia struthiopteris [L.] Todaro). Peak endopeptidase, aminopeptidase, and carboxypeptidase activities were detected 12 to 24 hours after spores began imbibing under light. There was a correlation between activities of proteases, the onset of a decline in levels of soluble protein, and an increase in levels of free amino acids. The earliest visible event of spore germination, breakage of the spore coat and protrusion of a rhizoid cell, was observed after peak protease activity, 48 to 72 hours after the start of imbibition. Results of this study demonstrate similarities in the pattern of protease activities during germination of ostrich fern spores to those of some seeds.     

Pyruvate metabolism in germinating seeds during natural anaerobiosis

Lactate as well as ethanol is formed in seeds of soybean, maize, pea, bean, lentil and broad-bean in the course of germination during the so-called natural anaerobiosis. After 0 to 30 h of germination a concentration peak of lactate appears. Maximum in ethanol content is found after 40 h. The amount of ethanol is higher big more than one order of magnitude as compared to the amount of lactate. Both products of anaerobiosis occur in germinating seeds irrespective of the type of reserve substances. In contrast to alcohol dehydrogenase lactate dehydrogenase (EC 1. 1. 1. 27) is present in the dry seeds too. Its activity decreases during the first 12 h of germination. It is in this stage that its substrate, lactate, is usually present at a maximal concentration. During the later stages of germination the amount of lactate decreases and enzyme activity rises. There exists a reciprocal relationship between enzyme activity and substrate concentration. In the case of alcohol dehydrogenases (EC 1. 1. 1. 1) the maximum concentration of ethanol precedes the peak of enzyme activity.     

High-Precision Fitting Measurements of the Kinetics of Size Changes during Germination of Individual Bacillus Spores

High-precision measurements of size changes of individual bacterial spores based on ellipse fitting to bright-field images recorded with a digital camera were employed to monitor the germination of Bacillus spores with a precision of ∼5 nm. To characterize the germination of individual spores, we recorded bright-field and phase-contrast images and found that the timing of changes in their normalized intensities coincided, so the bright-field images can be used to characterize spore size and refractility changes during germination. The major conclusions from this work were as follows. (i) The sizes of germinating B. cereus spores were nearly unchanged until T_release, the time of the completion of CaDPA (a 1:1 chelate of Ca²⁺ and dipicolinic acid [DPA]) release after addition of nutrient germinants. (ii) The minor axis of germinating B. cereus spores rapidly increased by ∼50 nm in a few seconds right after T_release, while the major axis was slightly decreased or unchanged. Both the minor and major axes remained unchanged for a further 30 to 45 s and then increased by 100 to 200 nm by T_lys, the time of completion of cortex lysis. (iii) Individual spores in a population showed significant heterogeneity in the timing of germination events, such as T_release and T_lys, but also variation in size changes during germination. (iv) Bacillus subtilis wild-type spores, B. subtilis spores lacking the cortex-lytic enzyme CwlJ, and wild-type Bacillus megaterium spores showed similar kinetics of size changes during nutrient germination. The size increases in germinating spores probably result from uptake of water and cortex lysis after completion of CaDPA release.     

Phytotoxic effects of fumonisin B1 on maize seedling growth

Fumonisin B₁ toxin is produced by the fungusFusarium moniliforme Sheldon, which is systemic to maize (Zea mays L.) and maize seeds. The effects of zero to 100 parts per million fumonisin B₁ on the germination process of maize seeds was determined. The presence of fumonisin had no effect on percent seed germination, but fumonisin inhibited radicle elongation by up to 75% after 48 hours of imbibition. An analysis of amylase secretion in the maize endosperm indicated that fumonisins inhibited amylase production in the germinating seed. Isoelectric focusing of endosperm extracts indicated that secretion of the low pI class of amylases was affected more that other amylase isozymes. The results suggested that the presence of high levels of fumonisin in maize seed may have deleterious effects on seedling emergence.     

Expression of the gene encoding the PR-like protein PRms in germinating maize embryos

Summary The PRms protein is a pathogenesis-related (PR)-like protein whose mRNA accumulates during germination of maize seeds. Expression of the PRms gene is induced after infection of maize seeds with the fungus Fusarium moniliforme. To further our investigations on the expression of the PRms gene we examined the accumulation of PRms mRNA in different tissues of maize seedlings infected with E. moniliforme and studied the effect of fungal elicitors, the mycotoxin moniliformin, the hormone gibberellic acid, and specific chemical agents. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by F. monilforme, increase the steady-state level of PRms mRNA. PRms mRNA accumulation is also stimulated by the application of the hormone gibberellic acid or by treatment with silver nitrate, whereas acetylsalicylic acid has no effect. In situ RNA hybridization in isolated germinating embryo sections demonstrates that the PRms gene is expressed in the scutellum, particularly in a group of inner cells, and in the epithelium lying at the interface of the scutellum and the endosperm. The pattern of expression of the PRms gene closely resembles that found for hydrolytic enzymes, being confined to the scutellum and the aleurone layer of the germinating maize seed. Our results suggest that the PRms protein has a function during the normal process of seed germination that has become adapted to serve among the defence mechanisms induced in response to pathogens during maize seed germination.     

Analysis of Proteome Profile in Germinating Soybean Seed,and Its Comparison with Rice Showing the Styles of Reserves Mobilization in Different Crops

Background

Seed germination is a complex physiological process during which mobilization of nutrient reserves happens. In different crops, this event might be mediated by different regulatory and metabolic pathways. Proteome profiling has been proved to be an efficient way that can help us to construct these pathways. However, no such studies have been performed in soybean germinating seeds up to date.

Results

Proteome profiling was conducted through one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry strategy in the germinating seeds of soybean (glycine max). Comprehensive comparisons were also carried out between rice and soybean germinating seeds. 764 proteins belonging to 14 functional groups were identified and metabolism related proteins were the largest group. Deep analyses of the proteins and pathways showed that lipids were degraded through lipoxygenase dependent pathway and proteins were degraded through both protease and 26S proteosome system, and the lipoxygenase could also help to remove the reactive oxygen species during the rapid mobilization of reserves of soybean germinating seeds. The differences between rice and soybean germinating seeds proteome profiles indicate that each crop species has distinct mechanism for reserves mobilization during germination. Different reserves could be converted into starches before they are totally utilized during the germination in different crops seeds.

Conclusions

This study is the first comprehensive analysis of proteome profile in germinating soybean seeds to date. The data presented in this paper will improve our understanding of the physiological and biochemical status in the imbibed soybean seeds just prior to germination. Comparison of the protein profile with that of germinating rice seeds gives us new insights on mobilization of nutrient reserves during the germination of crops seeds.     

A Method for Extracting RNA from Dormant and Germinating Bacillus subtilis Strain 168 Endospores

RNA was extracted from dormant and germinating Bacillus subtilis 168 spores (intact spores and chemically decoated spores) by using rapid rupture followed by acid–phenol extraction. Spore germination progress was monitored by assaying colony forming ability before and after heat shock and by reading the optical density at 600 nm. The purity, yield, and composition of the extracted RNA were determined spectrophotometrically from the ratio of absorption at 260 nm to that at 280 nm; in a 2100 BioAnalyzer, giving the RNA yield/10⁸ spores or cells and the distribution pattern of rRNA components. The method reported here for the extraction of RNA from dormant spores, as well as during different phases of germination and outgrowth, has proven to be fast, efficient and simple to handle. RNA of a high purity was obtained from dormant spores and during all phases of germination and growth. There was a significant increase in RNA yield during the transition from dormant spores to germination and subsequent outgrowth. Chemically decoated spores were retarded in germination and outgrowth compared with intact spores, and less RNA was extracted; however, the differences were not significant. This method for RNA isolation of dormant, germinating, and outgrowing bacterial endospores is a valuable prerequisite for gene expression studies, especially in studies on the responses of spores to hostile environmental conditions.     

The effect of leaf exudates on the spore germination of phylloplane mycoflora of chilli (Capsicum annuum L.) cultivars

Summary Ninetten aminoacids, twelve sugars, eleven organic acids and ten phenols were detected in the leaf exudates of three cultivars of chilli. The number of aminoacids, sugars, organic acids and phenols increased as the plants grew older. More aminoacids and sugars were detected in the exudate from the susceptible cultivar (Malwa). More organic acids and phenols were detected from the resistant cultivar (Simla). The leaf exudate of the resistant cultivar (Simla) inhibited spore germination of the pathogen (Alternaria solani) while that of susceptible (Malwa) stimulated spore germination. The cultivar ‘Patna’ which is moderately resistant, occupied an intermediate position. Spore germination of the isolated fungi was enhanced in leaf exudate of susceptible cultivar (Malwa), while leaf exudates of the moderately resistant (Patna) and resistant (Simla) inhibited spore germination of the majority of fungi isolated. Most of the antagonistic fungi were not isolated from the susceptible cultivar and the percentage spore germination of these fungi was less in leaf exudate of the susceptible cultivar, while leaf exudates of resistant cultivars enhanced the percentage spore germination of antagonistic fungi,viz Aspergillus flavus, A. fumigatus, A. versicolor, Penicillium citrinum, P. restrictum andTrichoderma viride.     

Differential sensitivity to cadmium in germinating seeds of three cultivars of faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.)

The reserve mobilization was analysed in germinating seeds of faba bean (Vicia faba) exposed to treatment with a toxic cadmium concentration for 4 days. When the behaviours of three cultivars were compared with regard to the germination rate, the following order of sensitivity to cadmium was observed: Aguadulce and Luz de otoño showed 59 and 19% of inhibition from controls, respectively, while no effect was observed in the case of the local Féverole. The growth of embryo radicle was also affected in the same pattern. The differential vulnerability to Cd stress cannot be correlated to shortage in water supply of cotyledons. However, Cd-treated germinating seeds of the most sensitive cultivar (Aguadulce) showed restriction in starch mobilization and decrease in availability of soluble sugars and free amino acids. Moreover, glucose, fructose and amino acids were markedly leaked into the germination medium at the expense of the growing embryonic axis during exposure to Cd. These results provide an indication of the way in which cadmium might impair seed germination.