Delay In Germination Research Articles

A significant correlation between ABA-induced seed-germination delay and salt tolerance of seedling in Brassica napus

ABSTRACT Under abiotic stresses, such as salt stress, abscisic acid (ABA) production is induced and enhances stress tolerance. ABA is also an important plant hormone that regulates vital biological responses, including seed gemination delay. However, the interplay between stress conditions and biological responses is not well understood. Previously, we reported a wide salt tolerance variation among Brassica napus varieties. This range of salt tolerance makes B. napus ideal for exploring the relationship between salt tolerance and ABA-induced seed germination delay. First, we investigated the ABA-induced seed-germination delay of 22 B. napus varieties, each with different salt tolerances. We observed that highly salt-tolerant varieties exhibited significant seed-germination delays when exposed to increasing ABA concentrations. Additionally, among the varieties, exogenous ABA induced a range of different germination delays, suggesting a significant correlation between germination delay and salt tolerance at the seedling stage. Expression analysis, with and without ABA treatment, for several genes involved in the ABA signalling pathway and downstream was also performed at seed germination. The results suggest the potential involvement of ABI5 and SOS1 in both germination regulation and salt tolerance.

Molecular mechanism of vivipary as revealed by the genomes of viviparous mangroves and non-viviparous relatives

Vivipary is a prominent feature of mangroves, allowing seeds to complete germination while attached to the mother plant, and equips propagules to endure and flourish in challenging coastal intertidal wetlands. However, vivipary-associated genetic mechanisms remain largely elusive. Genomes of two viviparous mangrove species and a non-viviparous inland relative were sequenced and assembled at the chromosome level. Comparative genomic analyses between viviparous and non-viviparous genomes revealed that DELAY OF GERMINATION 1 (DOG1) family genes (DFGs), the proteins from which are crucial for seed dormancy, germination, and reserve accumulation, are either lost or dysfunctional in the entire lineage of true viviparous mangroves but are present and functional in their inland, non-viviparous relatives. Transcriptome dynamics at key stages of vivipary further highlighted the roles of phytohormonal homeostasis, proteins stored in mature seeds, and proanthocyanidins in vivipary under conditions lacking DFGs. Population genomic analyses elucidate dynamics of syntenic regions surrounding the missing DFGs. Our findings demonstrated the genetic foundation of constitutive vivipary in Rhizophoraceae mangroves.

NaHS immersion alleviates the stress effect of chromium(III) on alfalfa seeds by affecting active oxygen metabolism

ABSTRACT Objective This study aimed to investigate the regulatory effects of exogenous hydrogen sulfide (H2S) on seed germination, seedling growth, and reactive oxygen species (ROS) homeostasis in alfalfa under chromium (Cr) ion (III) stress. Methods The effects of 0–4 mM Cr(III) on the germination and seedling growth of alfalfa were first assessed. Subsequently, following seed NaHS immersion, the influence of H2S on alfalfa seed germination and seedling growth under 2 mM Cr(III) stress was investigated, and the substance contents and enzyme activities associated with ROS metabolism were quantified. Results Compared to the control group, alfalfa plant germination was delayed under 2 mM Cr(III) stress for up to 48 h (p < 0.05). At 120 h, the total seedling length was approximately halved, and the root length was roughly one-third of the control. Treatment with 0.02–0.1 mM NaHS alleviated the delay in germination and root growth inhibition caused by 2 mM Cr(III) stress, resulting in an increased ratio of root length to hypocotyl length from 0.57 to 1 above. Additionally, immersion in 0.05 mM NaHS reduced hydrogen peroxide (H2O2) and oxygen-free radicals (O2 · –) levels (p < 0.05), boosted glutathione (GSH) levels (p < 0.05), and notably enhanced catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) activities (p < 0.05) compared to the 2 mM Cr(III) stress treatment group. Conclusion Seed immersion in NaHS mitigated the delay in germination and inhibition of root elongation under 2 mM Cr(III) stress. This effect is likely attributed to the regulation of intracellular ROS homeostasis and redox balance through enzymatic and non-enzymatic systems; thus, providing a potential mechanism for combating oxidative stress.

Arabidopsis DNA glycosylase MBD4L improves recovery of aged seeds.

DNA glycosylases initiate the base excision repair (BER) pathway by catalyzing the removal of damaged or mismatched bases from DNA. The Arabidopsis DNA glycosylase methyl-CpG-binding domain protein 4 like (MBD4L) is a nuclear enzyme triggering BER in response to the genotoxic agents 5-fluorouracil and 5-bromouracil. To date, the involvement of MBD4L in plant physiological processes has not been analyzed. To address this, we studied the enzyme functions in seeds. We found that imbibition induced the MBD4L gene expression by generating two alternative transcripts, MBD4L.3 and MBD4L.4. Gene activation was stronger in aged than in non-aged seeds. Seeds from mbd4l-1 mutants displayed germination failures when maintained under control or ageing conditions, while 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 seeds reversed these phenotypes. Seed nuclear DNA repair, assessed by comet assays, was exacerbated in an MBD4L-dependent manner at 24 h post-imbibition. Under this condition, the BER genes ARP, APE1L, and LIG1 showed higher expression in 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 than in mbd4l-1 seeds, suggesting that these components could coordinate with MBD4L to repair damaged DNA bases in seeds. Interestingly, the ATM, ATR, BRCA1, RAD51, and WEE1 genes associated with the DNA damage response (DDR) pathway were activated in mbd4l-1, but not in 35S:MBD4L.3/mbd4l-1 or 35S:MBD4L.4/mbd4l-1 seeds. These results indicate that MBD4L is a key enzyme of a BER cascade that operates during seed imbibition, whose deficiency would cause genomic damage detected by DDR, generating a delay or reduction in germination.

Histone modifications affecting plant dormancy and dormancy release: common regulatory effects on hormone metabolism.

As sessile organisms, plants enter periods of dormancy in response to environmental stresses to ensure continued growth and reproduction in the future. During dormancy, plant growth is suppressed, adaptive/survival mechanisms are exerted, and stress tolerance increases over a prolonged period until the plants resume their development or reproduction under favorable conditions. In this review, we focus on seed dormancy and bud dormancy, which are critical for adaptation to fluctuating environmental conditions. We provide an overview of the physiological characteristics of both types of dormancy as well as the importance of the phytohormones abscisic acid and gibberellin for establishing and releasing dormancy, respectively. Additionally, recent epigenetic analyses have revealed that dormancy establishment and release are associated with the removal and deposition of histone modifications at the loci of key regulatory genes influencing phytohormone metabolism and signaling, including DELAY OF GERMINATION 1 and DORMANCY-ASSOCIATED MADS-box genes. We discuss our current understanding of the physiological and molecular mechanisms required to establish and release seed dormancy and bud dormancy, while also describing how environmental conditions control dormancy depth, with a focus on the effects of histone modifications.

The significance of the two cytosolic glutamine synthetase enzymes, GLN1;3 and GLN1;5, in the context of seed development and germination in Arabidopsis thaliana

Glutamine synthetase (GS), an initial enzyme in nitrogen (N) plant metabolism, exists as a group of isoenzymes found in both cytosolic (GS1) and plastids (GS2) and has gathered significant attention for enhancing N use efficiency and crop yield.This work focuses on the A. thaliana GLN1;3 and GLN1;5 genes, the two predicted most expressed genes in seeds, among the five isogenes encoding GS1 in this species. The expression patterns were studied using transgenic marker line plants and qPCR during seed development and germination. The observed patterns highlight distinct functions for the two genes and confirm GLN1;5 as the most highly expressed GS1 gene in seeds. The GLN1;5, expression, oriented towards hypocotyl and cotyledons, suggests a role in protein turnover during germination, while the radicle-oriented expression of GLN1;3 supports a function in early external N uptake.While the single mutants exhibited a normal phenotype, except for a decrease in seed parameters, the double gln1;3/gln1;5 mutant displayed a germination delay, substantial impairment in growth, nitrogen metabolism, and number and quality of the seeds, as well as a diminishing in flowering. Although seed and pollen-specific, GLN1;5 expression is upregulated in the meristems of the gln1;3 mutants, filling the lack of GLN1;3 and ensuring the normal functioning of the gln1;3 mutants.These findings validate earlier in silico data on the expression patterns of GLN1;3 and GL1;5 genes in seeds, explore their different functions, and underscore their essential role in plant growth, seed production, germination, and early stages of plant development.

Effects of Orange Waste Extract Produced by Hydrodynamic Cavitation on the Germination of Chenopodium album L. and Lactuca sativa L.

A byproduct from orange juice processing known as pastazzo represents a significant organic waste stream. Rich in essential oils and known for its inhibitory effect on plant germination, pastazzo could serve as a valuable input for agricultural purposes. This study assesses the effects of a 40% w/v orange pastazzo water extract (OPWE) produced by hydrodynamic cavitation on the germination of two species, one of economic interest (Lactuca sativa L.) and one common weed (Chenopodium album L.). Three dilutions of OPWE in water (25%; 50%; 75%) were compared to a control treatment in four experiments, using (i) seeds in Petri dishes; (ii) seeds in commercial substrate; (iii) C. album seeds and transplanted L. sativa in commercial substrate; and (iv) other weeds in an open-field plantation of L. sativa. Highly rich in limonene, OPWE applied at higher concentrations in Petri dishes caused the effective inhibition of germination in C. album and a germination delay in L. sativa. Similar results were observed in the germination of the two species in commercial substrate, with none of the dilutions affecting L. sativa biomass. In the field experiment, despite a relatively low number of weeds in the control treatment, higher OPWE concentrations reduced the number of grasses and forbs, largely confirming the inhibitory effects. We conclude that OPWE produced with hydrodynamic cavitation, an efficient and affordable method of extraction, represents an effective crop treatment due to the species-specific effects of its constituent limonene on plant germination. Further tests are essential to understand the extent to which OPWE interacts with other species and types of substrate.

FsCGBP, a Cutinase G-Box Binding Protein, Regulates the Growth, Development, and Virulence of Fusarium sacchari, the Pathogen of Sugarcane Pokkah Boeng Disease.

Fusarium sacchari is a causal agent of sugarcane Pokkah boeng, an important fungal disease that causes a considerable reduction in yield and sugar content in susceptible varieties of sugarcane worldwide. Despite its importance, the fungal factors that regulate the virulence of this pathogen remain largely unknown. In our previous study, mapping of an insertional mutant defect in virulence resulted in the identification of a cutinase G-box binding protein gene, designated FsCGBP, that encodes a C2H2-type transcription factor (TF). FsCGBP was shown to localize in the nuclei, and the transcript level of FsCGBP was significantly upregulated during the infection process or in response to abiotic stresses. Deletion or silencing of FsCGBP resulted in a reduction in mycelial growth, conidial production, and virulence and a delay in conidial germination in the F. sacchari. Cutinase genes FsCUT2, FsCUT3, and FsCUT4 and the mitogen-activated protein kinase (MAPK) genes FsHOG1, FsMGV1, and FsGPMK1, which were significantly downregulated in ΔFsCGBP. Except for FsHOG1, all of these genes were found to be transcriptionally activated by FsCGBP using the yeast one-hybrid system in vitro. The deletion of individual cutinase genes did not result in any of the phenotypes exhibited in the ΔFsCGBP mutant, except for cutinase activity. However, disruption of the MAPK pathway upon deletion of FsMGV1 or FsGPMK1 resulted in phenotypes similar to those of the ΔFsCGBP mutant. The above results suggest that FsCGBP functions by regulating the MAPK pathway and cutinase genes, providing new insights into the mechanism of virulence regulation in F. sacchari.

The Effect of Heat Shock on Seed Dormancy Release and Germination in Two Rare and Endangered Astragalus L. Species (Fabaceae).

Many Astragalus species exhibit seeds with physical dormancy (PY), but little is known about the ecological context of this dormancy. We focused on A. maritimus and A. verrucosus, two threatened Sardinian endemic species inside the subgenus Trimeniaeus Bunge. Fresh seeds collected from the only two respective known populations were used to investigate the effect of mechanical scarification, heat shock, and water imbibition processes on PY release and germination. PY can be overcome through mechanical scarification of the water-impermeable seed coats, while no dormancy break was detected, nor a subsequent increase in seed germination due to fire-induced heat. This suggests that fire does not trigger dormancy release and seed germination in these species. The seeds tolerate relatively high heat shock temperatures (up to 120 and 100 °C for A. verrucosus and A. maritimus, respectively), but after 120 °C for 10 min, the number of dead seeds increases in both species. These facts suggest the capacity to develop a soil seed bank that can persist after fires and delay germination until the occurrence of optimal conditions. As regards water imbibition, both Astragalus species did not show the typical triphasic pattern, as germination started without further water uptake. This study emphasizes the significance of understanding germination processes and dormancy in threatened species. In fire-prone ecosystems, PY dormancy plays a crucial role in soil seed bank persistence, and it may be selectively influenced by post-fire conditions. Understanding such adaptations provides useful insights into conservation strategies.

Proteomic analysis during seed development provides insight into the early establishment of seed dormancy in Xanthium strumarium.

This experiment was carried out to provide a comprehensive insight into the protein activities involved in dormancy establishment in seeds of common cocklebur (Xanthium strumarium), an annual plant with two dimorphic seeds contained in one casing known as a burr. These consist of a smaller dormant seed and a larger non-dormant seed. The proteome profile was compared between developing dormant and non-dormant seeds of Xanthium strumarium at five consecutive stages including three, 10, 20, 30, and 45 days after burr emergence (stages 1 to 5). We identified 6524 proteins in total, and approximately 3.6% of these were differentially abundant proteins (DAPs) between the two seed types. Both seed types showed fundamental changes in developmental programs during the examined stages. More than 38% of all DAPs were observed at the first stage, supporting the importance of the early developmental stage in seed fate determination. The detected DAPs at stage 1 were mainly associated with the cell division phase, which showed a delay in the dormant seeds. Over-representation of proteins responsible for cell wall biosynthesis, cytokinesis, and seed development were detected for non-dormant seeds at the first stage, while dormancy-associated proteins showed less abundance. Stage 3 was the critical stage for switching processes toward seed maturation and abscisic acid (ABA) signaling. Interestingly, higher abundance proteins in the mature non-dormant seed were mainly involved in the facilitation of seed germination. Taken together, the temporal pattern of the accumulated proteins in developing dormant seeds demonstrated a delay in the initiation of active cell division, enriched response to ABA, and defects in seed maturation. Moreover, stored proteins in the mature dormant seed delay germination but not dormancy induction. Finally, our results suggest that dormancy may be established at a stage of seed development earlier than previously thought.

Impact of sporulation temperature on germination of Bacillus subtilis spores under optimal and adverse environmental conditions

Bacillus subtilis spores are important food spoilage agents and are occasionally involved in food poisoning. In foods that are not processed with intense heat, such bacterial spores are controlled by a combination of different hurdles, such as refrigeration, acidification, and low water activity (aw), which inhibit or delay germination and/or growth. Sporulation temperature has long been regarded as a relevant factor for the assessment of germination in chemically defined media, but little is known about its impact on food preservation environments. In this study, we compared germination dynamics of B. subtilis spores produced at optimal temperature (37 °C) with others incubated at suboptimal (20 °C) and supraoptimal (43 °C) temperatures in a variety of nutrients (rich-growth medium, L-alanine, L-valine, and AGFK) under optimal conditions as well as under food-related stresses (low aw, pH, and temperature). Spores produced at 20 °C had a lower germination rate and efficiency than those incubated at 37 °C in all the nutrients, while those sporulated at 43 °C displayed a higher germination rate and/or efficiency in response to rich-growth medium and mostly to L-alanine and AGFK under optimal environmental conditions. However, differences in germination induced by changes in sporulation temperature decreased when spores were activated by heat, mainly due to the greater benefit of heat for spores produced at 20 °C and 37 °C than at 43 °C, especially in AGFK. Non-heat-activated spores produced at 43 °C still displayed superior germination fitness under certain stresses that had considerably impaired the germination of the other two populations, such as reduced temperature and aw. Moreover, they presented lower temperature and pH boundaries for the inhibition of germination in rich-growth medium, while requiring a higher NaCl concentration threshold compared to spores obtained at optimal and suboptimal temperature. Sporulation temperature is therefore a relevant source of variability in spore germination that should be taken into account for the accurate prediction of spore behaviour under variable food preservation conditions with the aim of improving food safety and stability.

Long term impacts of endozoochory and salinity on germination of wetland plants after entering simulated seed banks.

Migratory waterbirds disperse a broad range of angiosperms by endozoochory (seed dispersal via gut passage), especially plants in coastal wetlands. However, there is no previous information about the capacity of seeds to remain in the seed bank after waterbird endozoochory, and very little about how wetland salinity can influence the effect of gut passage on germination. We collected seeds of Juncus subulatus (Juncaceae), Bolboschoenus maritimus, and Schoenoplectus litoralis (Cyperaceae) from Doñana marshes in Spain. All three species are considered to have physiological dormancy. After gut passage following ingestion by ducks, seeds were stored in darkness in solutions with six different conductivities (1, 2, 4, 8, 16, and 32 dSm-1), for periods of 1, 6, or 12 months to simulate presence in a seed bank. After storage, 1800 seeds of each plant species assigned to these treatments were subjected to germination tests in demineralized water, together with 1800 control seeds that had not been ingested before storage. All species germinated readily after storage, with or without gut passage beforehand. Storage time and salinity both had important effects on germinability and time to germination, which differed between control and ingested seeds, and between plant species. After ≥6 months, germinability of Cyperaceae was enhanced by gut passage (≤25% higher than control seeds) at some salinities. Only J. subulatus showed consistently lower germinability after passage (≤30%). Only B. maritimus showed consistently slower germination after passage (≤33%). Salinity effects were more complex after passage, but increasing salinity did not generally have a negative impact on germination of ingested seeds. When compared to additional seeds that had not been stored before germination tests, storage reduced germinability in J. subulatus (≤39% reduction), but increased it in B. maritimus (≤17%) and S. litoralis (≤46%). Seeds dispersed by waterbird endozoochory may be easily incorporated into wetland seed banks, where they can remain halotolerant and delay germination until conditions become suitable. This can benefit wetland plants by increasing rates of long-distance dispersal, gene flow, and establishment of new populations. Avian gut passage can have positive and species-specific effects on germination in plants with persistent seed banks and/or physiological dormancy.

Maturation indexes and physiological quality of seeds and fruits of Morinda citrifolia L.

The seeds of Morinda citrifolia L. present unevenness and delay in germination in relation to other fruit trees. Knowledge about the physiological quality of seeds is extremely relevant for the production of seedlings and propagation of the species. Thus, the objective was to study maturation indices and the physiological quality of seeds and fruits of M. citrifolia. Seeds from freshly picked fruits, fruits harvested after seven days of maturation and freshly dropped fruits were used. The extracted and processed seeds were submitted to the following pre-germination treatments: GA3, KNO3 and H2O. The following were analyzed: fruit length and diameter, seed length, width and thickness, number of seeds per fruit, average number of seeds per kg, weight of 1000 seeds, germination, germination speed index, shoot and root length, seedling dry mass and percentage of normal seedlings. The experimental design used was completely randomized, with three stages of fruit maturation and six treatments, with four replications of 25 seeds. M. citrifolia seeds do not show tegumentary dormancy. The seeds of freshly harvested fruits and fruits harvested after seven days of maturation do not need pre-germination treatments. GA3 promotes greater germination in seeds of fruits harvested from the ground. Fruits that have just fallen to the ground have a higher content of soluble solids and a higher content of acidity. The storage of fruit in a refrigerator for a week provides the maintenance of texture characteristics, such as hardness, adhesiveness, elasticity and gumminess. Fruits with greater hardness have higher physiological seed quality.

The role of micropyle for grass pea germination

PurposeThis study aims to identify the location of the micropyle, the role of the micropyle in seed germination and the association between the micropyle size and seed weight of grass peas.Design/methodology/approachFirst, the micropyle was identified by cutting the seed in half and observing the seeds under the electron microscope. Second, the micropyle was covered by lanolin to block water imbibition. The rate of imbibition and germination was then observed. Lastly, micropyle sizes of various grass pea genotypes were identified by capturing seed images under a light microscope and converting the sizes to mm2 using computer software (ImageJ).FindingsThe location of micropyle was located nearby the hilum, similar to soybean seeds. Seed imbibition was significantly lower in lanolin application (&lt;87%) than in the control (&gt;124%) after 24 hours of submergence. Germination was a day delay for lanolin application on the micropyle compared to lanolin application on the non-micropyle. The germination delay resulted in a significantly lower germination percentage at &lt;57% on the micropyle lanolin application than at &gt;79% on the non-micropyle lanolin application after 10 days of sowing. There is no correlation between the micropyle size and seed weight.Originality/valueThese findings add information on the location and the role of the micropyle for grass pea seed germination.

Arabidopsis histone deacetylase HD2A and HD2B regulate seed dormancy by repressing DELAY OF GERMINATION 1.

Seed dormancy is a crucial developmental transition that affects the adaption and survival of plants. Arabidopsis DELAY OF GERMINATION 1 (DOG1) is known as a master regulator of seed dormancy. However, although several upstream factors of DOG1 have been reported, the exact regulation of DOG1 is not fully understood. Histone acetylation is an important regulatory layer, controlled by histone acetyltransferases and histone deacetylases. Histone acetylation strongly correlates with transcriptionally active chromatin, whereas heterochromatin is generally characterized by hypoacetylated histones. Here we describe that loss of function of two plant-specific histone deacetylases, HD2A and HD2B, resulted in enhanced seed dormancy in Arabidopsis. Interestingly, the silencing of HD2A and HD2B caused hyperacetylation of the DOG1 locus and promoted the expression of DOG1 during seed maturation and imbibition. Knockout of DOG1 could rescue the seed dormancy and partly rescue the disturbed development phenotype of hd2ahd2b. Transcriptomic analysis of the hd2ahd2b line shows that many genes involved in seed development were impaired. Moreover, we demonstrated that HSI2 and HSL1 interact with HD2A and HD2B. In sum, these results suggest that HSI2 and HSL1 might recruit HD2A and HD2B to DOG1 to negatively regulate DOG1 expression and to reduce seed dormancy, consequently, affecting seed development during seed maturation and promoting seed germination during imbibition.

Effect of Temperature on Breaking of Morphophysiological Dormancy and Seed Germination Leading to Bulblet Production in Two Endemic Tulip Species from Greece.

Due to botanical tulips' economic interest coupled with limited information regarding their seed germination, we investigated the effect of temperature on dormancy release and germination in two endangered local endemic tulip species of Greece (Tulipa hageri Heldr., T. orphanidea Heldr.). Their germination responses at five constant temperatures (5, 10, 15, 20, and 25 °C) were evaluated in growth chambers, while the type of seed dormancy and the temperature effect on its release were determined based on open-sourced, R-derived species-specific ecological profiles illustrating abiotic conditions of their wild habitats. The results indicated a range-specific temperature dependence in seed germination for both studied species with seed germination observed only in very low temperatures (5-10 °C). The seeds of both species after dispersal had an underdeveloped embryo. The existence of a complex morphophysiological seed dormancy was confirmed in both species by the significant embryo development only at 5 and 10 °C (almost doubled after 30 days) coupled with observed delay in germination only at low temperatures. Furthermore, to facilitate their cultivation and ex situ conservation, the germinated seeds were planted in pots to develop bulblets in greenhouse conditions resulting in bigger T. orphanidea bulblets compared to T. hageri.

EFFECTS OF TEMPERATURE VARIATIONS AND PRE-SOWING TREATMENTS ON THE GERMINATION OF Milicia excelsa: A CASE STUDY OF SEEDS COLLECTED FROM BENCHI-MAJI ZONE, SOUTH-WESTERN ETHIOPIA

Seeds always exhibit some degree of dormancy, resulting in a delay and irregularity of germination, and the seeds of Milicia excelsa often have seed germination difficulty. The main objective of this study was to demonstrate the seed germination variations of M. excelsa at room temperature (20-25°C) and incubator (25.6o C). In this study, 400 seeds of M. excelsa were tested using three pre-sowing treatments and control. Each treatment was defined as four replications in which 25 seeds per replication were initially sown in a 90 mm diameter petri dish with disc paper in a completely randomized design. The result showed that the mean germination percentage of the control treatment scored about 23.8%, followed by seeded rubbed by hand with a score of 15%. It is observed that room temperature has a significant effect (29%) on the germination of the control treatment compared to the incubator (18.7%) at p=0.05. However, no significant mean germination difference is observed between the effects of room temperature and the incubator in the remaining pre-sowing treatments: Washed with tap water (T1), 2) Washed and soaked in hot water for 15 minutes (T2) and 3) rubbing by hand (T3). However, it can be inferred that room temperature is an ideal temperature that meets plants' physiological seed germination requirement. To understand the importance of temperature and light on the germination of angiosperm, further experiments involving variable ranges of temperature and light intensity can be conducted.

Ada2 and Ada3 Regulate Hyphal Growth, Asexual Development, and Pathogenicity in Beauveria bassiana by Maintaining Gcn5 Acetyltransferase Activity.

The histone acetyltransferase (HAT) Gcn5 ortholog is essential for a variety of fungi. Here, we characterize the roles of Ada2 and Ada3, which are functionally linked to Gcn5, in the insect-pathogenic fungus Beauveria bassiana. Loss of Ada2 and Ada3 led to severe hyphal growth defects on rich and minimal media and drastic decreases in blastospore yield and conidiation capacity, with abnormal conidia-producing structures. ΔAda2 and ΔAda3 exhibited a delay in conidial germination and increased sensitivity to multiple chemical stresses and heat shock. Nearly all their pathogenicity was lost, and their ability to secrete extracellular enzymes, Pr1 proteases and chitinases for cuticle degradation was reduced. A yeast two-hybrid assay demonstrated that Ada2 binds to Ada3 and directly interacts with Gcn5, confirming the existence of a yeast-like Ada3-Ada2-Gcn5 HAT complex in this fungus. Additionally, deletion of the Ada genes reduced the activity of Gcn5, especially in the ΔAda2 strain, which was consistent with the acetylation level of histone H3 determined by Western blotting. These results illustrate the dependence of Gcn5 enzyme activity on Ada2 and Ada3 in fungal hyphal growth, asexual development, multiple stress responses, and pathogenicity in B. bassiana. IMPORTANCE The histone acetyltransferase Gcn5 ortholog contributes significantly to the growth and development of various fungi. In this study, we found that Ada2 and Ada3 have critical regulatory effects on Gcn5 enzyme activity and influence the acetylation of histone H3. Deletion of Ada2 or Ada3 decreased the fungal growth rate and asexual conidial yield and increased susceptibility to multiple stresses in Beauveria bassiana. Importantly, Ada genes are vital virulence factors, and their deletion caused the most virulence loss, mainly by inhibiting the activity of a series of hydrolytic enzymes and the dimorphic transition ability. These findings provide a new perspective on the function of the Gcn5 acetyltransferase complex in pathogens.

Biocontrol potential of wine yeasts against four grape phytopathogenic fungi disclosed by time-course monitoring of inhibitory activities.

Grapes' infection by phytopathogenic fungi may often lead to rot and impair the quality and safety of the final product. Due to the concerns associated with the extensive use of chemicals to control these fungi, including their toxicity for environment and human health, bio-based products are being highly preferred, as eco-friendlier and safer alternatives. Specifically, yeasts have shown to possess antagonistic activity against fungi, being promising for the formulation of new biocontrol products.In this work 397 wine yeasts, isolated from Portuguese wine regions, were studied for their biocontrol potential against common grapes phytopathogenic fungal genera: Aspergillus, Botrytis, Mucor and Penicillium. This set comprised strains affiliated to 32 species distributed among 20 genera. Time-course monitoring of mold growth was performed to assess the inhibitory activity resulting from either diffusible or volatile compounds produced by each yeast strain. All yeasts displayed antagonistic activity against at least one of the mold targets. Mucor was the most affected being strongly inhibited by 68% of the tested strains, followed by Botrytis (20%), Aspergillus (19%) and Penicillium (7%). More notably, the approach used allowed the detection of a wide array of yeast-induced mold response profiles encompassing, besides the decrease of mold growth, the inhibition or delay of spore germination and the complete arrest of mycelial extension, and even its stimulation at different phases. Each factor considered (taxonomic affiliation, mode of action and fungal target) as well as their interactions significantly affected the antagonistic activity of the yeast isolates. The highest inhibitions were mediated by volatile compounds. Total inhibition of Penicillium was achieved by a strain of Metschnikowia pulcherrima, while the best performing yeasts against Mucor, Aspergillus and Botrytis, belong to Lachancea thermotolerans, Hanseniaspora uvarum and Starmerella bacillaris, respectively. Notwithstanding the wide diversity of yeasts tested, only three strains were found to possess a broad spectrum of antagonistic activity, displaying strong or very strong inhibition against the four fungal targets tested. Our results confirm the potential of wine yeasts as biocontrol agents, while highlighting the need for the establishment of fit-for-purpose selection programs depending on the mold target, the timing, and the mode of application.

Knockdown of Bmp1 and Pls1 Virulence Genes by Exogenous Application of RNAi-Inducing dsRNA in Botrytis cinerea

Botrytis cinerea is a pathogen of wide agronomic and scientific importance partly due to its tendency to develop fungicide resistance. Recently, there has been great interest in the use of RNA interference as a control strategy against B. cinerea. In order to reduce the possible effects on non-target species, the sequence-dependent nature of RNAi can be used as an advantage to customize the design of dsRNA molecules. We selected two genes related to virulence: BcBmp1 (a MAP kinase essential for fungal pathogenesis) and BcPls1 (a tetraspanin related to appressorium penetration). After performing a prediction analysis of small interfering RNAs, dsRNAs of 344 (BcBmp1) and 413 (BcPls1) nucleotides were synthesized in vitro. We tested the effect of topical applications of dsRNAs, both in vitro by a fungal growth assay in microtiter plates and in vivo on artificially inoculated detached lettuce leaves. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination for BcBmp1, an evident growth retardation for BcPls1, and a strong reduction in necrotic lesions on lettuce leaves for both genes. Furthermore, a strongly reduced expression of the BcBmp1 and BcPls1 genes was observed in both in vitro and in vivo experiments, suggesting that these genes could be promising targets for the development of RNAi-based fungicides against B. cinerea.