Inhibition of the lemon brown rot causal agent Phytophthora citrophthora by low-toxicity compounds.

BACKGROUND: Phytophthora spp., soil-borne oomycetes, cause brown rot (BR) on postharvest lemons. The management of this disease is based on cultural practices and chemical control using inorganic salts of limited efficacy. In the search for new alternatives, the aim of this work was to evaluate the effect of low-toxicity compounds to inhibit the growth of P. citrophthora and to control BR disease on lemons. Sodium bicarbonate, potassium sorbate, polyhexamethylene guanidine, Ascophyllum nodosum extract and a formulation containing phosphite salts plus A. nodosum (P+An) were evaluated. RESULTS: All tested products inhibited mycelial growth, sporangia formation and zoospore germination of P. citrophthora in vitro. In postharvest applications on artificially inoculated lemons, only P+An exhibited a BR curative effect, with incidence reduction of around 60%. When this formulation was applied in field treatments, BR incidence was reduced by 40% on lemons harvested and inoculated up to 30 days post application. CONCLUSION: Our results demonstrate the in vitro direct anti-oomycete effect of low-toxicity compounds and the in vivo efficacy of P+An formulation to control BR, encouraging the incorporation of the latter in the management of citrus BR. © 2020 Society of Chemical Industry.

Polyhexamethylene guanidine as a fungicide, disinfectant and wound protector in lemons challenged with Penicillium digitatum.

Citrus green mold, a postharvest disease caused by Penicillium digitatum, provokes important economic losses on lemon production. Here, the effectiveness of polyhexamethylene guanidine (PHMG) to inhibit P. digitatum growth and to control green mold on artificially infected lemons was evaluated. At sublethal concentrations, PHMG inhibited conidia germination and infectivity (5 mg L-1), and mycelial growth (50 mg L-1). Viability of conidia was completely suppressed by treatment with 500 mg L-1 PHMG. In this condition, membrane integrity loss, cell wall disruption and ultrastructural alterations were detected, as well as conidia distortion, deformation and collapse. In artificially inoculated lemons, a 30 s-immersion in 500 mg L-1 PHMG completely inhibited green mold. PHMG also exhibited a high disinfectant activity, even in the presence of 1% organic matter, with a better performance than the standard NaClO disinfectant. In addition, 500 mg L-1 PHMG protected wounds against infection. Taken together, our results indicate that PHMG is a promising fungicide for the postharvest control of green mold in lemon packinghouses.

Antifungal activity of ß-carbolines on Penicillium digitatum and Botrytis cinerea.

ß-carbolines (ßCs) are alkaloids widely distributed in nature that have demonstrated antimicrobial properties. Here, we tested in vitro six ßCs against Penicillium digitatum and Botrytis cinerea, causal agents of postharvest diseases on fruit and vegetables. Full aromatic ßCs (harmine, harmol, norharmane and harmane) exhibited a marked inhibitory effect on conidia germination at concentrations between 0.5 and 1 mM, while dihydro-ßCs (harmalina and harmalol) only caused germination delay. Harmol showed the highest inhibitory effect on both fungal pathogens. After 24 h of exposure to 1 mM harmol, conidia revealed a severe cellular damage, exhibiting disorganized cytoplasm and thickened cell wall. Harmol antimicrobial effect was fungicidal on B. cinerea, while it was fungistatic on P. digitatum. Conidia membrane permeabilization was detected in treatments with harmol at sub-inhibitory and inhibitory concentrations, for both pathogens. In addition, residual infectivity of P. digitatum on lemons and B. cinerea on blueberries was significantly reduced after exposure to this alkaloid. It also inhibited mycelial growth, preventing sporulation at the highest concentration tested. These results indicate that harmol might be a promising candidate as a new antifungal molecule to control causal agents of fruit diseases.

Removal of pathogenic bacterial biofilms by combinations of oxidizing compounds.

Bacterial biofilms are commonly formed on medical devices and food processing surfaces. The antimicrobials used have limited efficacy against the biofilms; therefore, new strategies to prevent and remove these structures are needed. Here, the effectiveness of brief oxidative treatments, based on the combination of sodium hypochlorite (NaClO) and hydrogen peroxide (H2O2) in the presence of copper sulfate (CuSO4), were evaluated against bacterial laboratory strains and clinical isolates, both in planktonic and biofilm states. Simultaneous application of oxidants synergistically inactivated planktonic cells and prevented biofilm formation of laboratory Escherichia coli, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Staphylococcus aureus strains, as well as clinical isolates of Salmonella enterica subsp. enterica, Klebsiella oxytoca, and uropathogenic E. coli. In addition, preformed biofilms of E. coli C, Salmonella Typhimurium, K. pneumoniae, and Salmonella enterica exposed to treatments were removed by applying 12 mg/L NaClO, 0.1 mmol/L CuSO4, and 350 mmol/L H2O2 for 5 min. Klebsiella oxytoca and Staphylococcus aureus required a 5-fold increase in NaClO concentration, and the E. coli clinical isolate remained unremovable unless treatments were applied on biofilms formed within 24 h instead of 48 h. The application of treatments that last a few minutes using oxidizing compounds at low concentrations represents an interesting disinfection strategy against pathogens associated with medical and industrial settings.

Regulation of sporulation initiation by NprR and its signaling peptide NprRB: molecular recognition and conformational changes.

NprR belongs to the RNPP family of quorum-sensing receptors, a group of intracellular regulators activated directly by signaling oligopeptides in Gram-positive bacteria. In Bacillus thuringiensis (Bt), nprR is located in a transcriptional cassette with nprRB that codes for the precursor of the signaling peptide NprRB. NprR is a transcriptional regulator activated by binding of reimported NprRB; however, several reports suggest that NprR also participates in sporulation but the mechanism is unknown. Our in silico results, based on the structural similarity between NprR from Bt and Spo0F-binding Rap proteins from Bacillus subtilis, suggested that NprR could bind Spo0F to modulate the sporulation phosphorelay in Bt. Deletion of nprR-nprRB cassette from Bt caused a delay in sporulation and defective trigger of the Spo0A∼P-activated genes spoIIA and spoIIIG. The DNA-binding domain of NprR was not necessary for this second function, since truncated NprRΔHTH together with nprRB gene was able to restore the sporulation wild type phenotype in the ΔnprR-nprRB mutant. Fluorescence assays showed direct binding between NprR and Spo0F, supporting that NprR is a bifunctional protein. To understand how the NprR activation by NprRB could result in two different functions, we studied the molecular recognition mechanism between the signaling peptide and the receptor. Using synthetic variants of NprRB, we found that SSKPDIVG displayed the highest affinity (Kd = 7.19 nM) toward the recombinant NprR and demonstrated that recognition involves conformational selection. We propose that the peptide concentration in the cell controls the oligomerization state of the NprR-NprRB complex for switching between its two functions.

Chemical and genetic basis of orange flavor.

Sweet orange (Citrus sinensis) exhibits limited genetic diversity and high susceptibility to Huanglongbing (HLB). Breeding HLB-tolerant orange-like hybrids is in dire need. However, our understanding of the key compounds responsible for orange flavor and their genetic regulation remains elusive. Evaluating 179 juice samples, including oranges, mandarins, Poncirus trifoliata, and hybrids, distinct volatile compositions were found. A random forest model predicted untrained samples with 78% accuracy and identified 26 compounds crucial for orange flavor. Notably, seven esters differentiated orange from mandarin flavor. Cluster analysis showed six esters with shared genetic control. Differential gene expression analysis identified C. sinensis alcohol acyltransferase 1 (CsAAT1) responsible for ester production in orange. Its activity was validated through overexpression assays. Phylogeny revealed the functional allele was inherited from pummelo. A SNP-based DNA marker in the coding region accurately predicted phenotypes. This study enhances our understanding of orange flavor compounds and their biosynthetic pathways and expands breeding options for orange-like cultivars.

Overcoming lemon postharvest molds caused by Penicillium spp. multiresistant isolates by the application of potassium sorbate in aqueous and wax treatments.

Penicillium digitatum and Penicillium italicum are the main causal agents of postharvest diseases in lemon. Over the last decades, the appearance of isolates resistant to the main commercial fungicides has been considered one of the most serious problems for the citrus industry. In this work, potassium sorbate (KS) was evaluated as an alternative to chemical fungicides to control postharvest diseases caused by Penicillium isolates resistant to imazalil, thiabendazol, and pyrimethanil. In vitro assays showed that 1% KS inhibited conidia germination and mycelial growth of sensitive and resistant P. digitatum and P. italicum isolates, being this effect stronger at pH 5 than at pH 9. In curative treatments, the immersion of inoculated lemons in 1% KS aqueous solution for 30 s reduced green and blue molds incidences by around 80%. No wound protection effect was observed when wounded lemons were immersed in 3% salt solution before inoculation. Noteworthy, the inclusion of KS in a commercial wax coating effectively controlled green and blue molds, even in decays caused by fungicide resistance isolates. Together, results encourage the use of KS in lemon postharvest treatments to contribute to the management of resistant strains, which represent a major challenge in packinghouses worldwide. PRACTICAL APPLICATION: The use of KS in citrus postharvest treatments would help producers to reduce spoilage caused by Penicillium fungicide-resistant strains. The inclusion of this generally recognized as safe compound in wax coatings improves its persistence on the fruit surface, keeping product quality during long-term overseas transport. In sum, KS constitutes an affordable and eco-friendly option for controlling postharvest molds in lemon fruit.

Application of Thymol Vapors to Control Postharvest Decay Caused by Penicillium digitatum and Lasiodiplodia theobromae in Grapefruit.

Two of the major postharvest diseases impacting grapefruit shelf life and marketability in the state of Florida (USA) are stem-end rot (SER) caused by Lasiodiplodia theobromae and green mold (GM) caused by Penicillium digitatum. Here, we investigated the in vitro and in vivo efficacy of vapors of thymol, a natural compound found in the essential oil of various plants and the primary constituent of thyme (Thymus vulgaris) oil, as a potential solution for the management of GM and SER. Thymol vapors at concentrations lower than 10 mg L-1 significantly inhibited the mycelial growth of both pathogens, causing severe ultrastructural damage to P. digitatum conidia. In in vivo trials, the incidence and lesion area of GM and SER on inoculated grapefruit were significantly reduced after a 5 d exposure to 50 mg L-1 thymol vapors. In addition, the in vitro and in vivo sporulation of P. digitatum was suppressed by thymol. When applied in its vapor phase, thymol had no negative effect on the fruit, neither introducing perceivable off-flavor nor causing additional weight loss. Our findings support the pursuit of further studies on the use of thymol, recognized as safe for human health and the environment, as a promising strategy for grapefruit postharvest disease management.

First draft genome sequence of a strain from the genus Citricoccus.

Bacteria of the genus Citricoccus have been isolated from ecological niches characterized by diverse abiotic stress conditions. Here we report the first genome draft of a strain of the genus Citricoccus isolated from the extremely oligotrophic Churince system in the Cuatro Ciénegas Basin (CCB) in Coahuila, Mexico.

The genome of Bacillus coahuilensis reveals adaptations essential for survival in the relic of an ancient marine environment.

The Cuatro Ciénegas Basin (CCB) in the central part of the Chihuahan desert (Coahuila, Mexico) hosts a wide diversity of microorganisms contained within springs thought to be geomorphological relics of an ancient sea. A major question remaining to be answered is whether bacteria from CCB are ancient marine bacteria that adapted to an oligotrophic system poor in NaCl, rich in sulfates, and with extremely low phosphorus levels (<0.3 microM). Here, we report the complete genome sequence of Bacillus coahuilensis, a sporulating bacterium isolated from the water column of a desiccation lagoon in CCB. At 3.35 Megabases this is the smallest genome sequenced to date of a Bacillus species and provides insights into the origin, evolution, and adaptation of B. coahuilensis to the CCB environment. We propose that the size and complexity of the B. coahuilensis genome reflects the adaptation of an ancient marine bacterium to a novel environment, providing support to a "marine isolation origin hypothesis" that is consistent with the geology of CCB. This genomic adaptation includes the acquisition through horizontal gene transfer of genes involved in phosphorous utilization efficiency and adaptation to high-light environments. The B. coahuilensis genome sequence also revealed important ecological features of the bacterial community in CCB and offers opportunities for a unique glimpse of a microbe-dominated world last seen in the Precambrian.

Understanding the evolutionary relationships and major traits of Bacillus through comparative genomics.

BACKGROUND: The presence of Bacillus in very diverse environments reflects the versatile metabolic capabilities of a widely distributed genus. Traditional phylogenetic analysis based on limited gene sampling is not adequate for resolving the genus evolutionary relationships. By distinguishing between core and pan-genome, we determined the evolutionary and functional relationships of known Bacillus. RESULTS: Our analysis is based upon twenty complete and draft Bacillus genomes, including a newly sequenced Bacillus isolate from an aquatic environment that we report for the first time here. Using a core genome, we were able to determine the phylogeny of known Bacilli, including aquatic strains whose position in the phylogenetic tree could not be unambiguously determined in the past. Using the pan-genome from the sequenced Bacillus, we identified functional differences, such as carbohydrate utilization and genes involved in signal transduction, which distinguished the taxonomic groups. We also assessed the genetic architecture of the defining traits of Bacillus, such as sporulation and competence, and showed that less than one third of the B. subtilis genes are conserved across other Bacilli. Most variation was shown to occur in genes that are needed to respond to environmental cues, suggesting that Bacilli have genetically specialized to allow for the occupation of diverse habitats and niches. CONCLUSIONS: The aquatic Bacilli are defined here for the first time as a group through the phylogenetic analysis of 814 genes that comprise the core genome. Our data distinguished between genomic components, especially core vs. pan-genome to provide insight into phylogeny and function that would otherwise be difficult to achieve. A phylogeny may mask the diversity of functions, which we tried to uncover in our approach. The diversity of sporulation and competence genes across the Bacilli was unexpected based on previous studies of the B. subtilis model alone. The challenge of uncovering the novelties and variations among genes of the non-subtilis groups still remains. This task will be best accomplished by directing efforts toward understanding phylogenetic groups with similar ecological niches.

Mini-III, a fourth class of RNase III catalyses maturation of the Bacillus subtilis 23S ribosomal RNA.

Ribonuclease III (RNase III) type of enzymes are double-stranded RNA (dsRNA)-specific endoribonucleases that have important roles in RNA maturation and mRNA decay. They are involved in processing precursors of ribosomal RNA (rRNA) in bacteria as well as precursors of short interfering RNAs (siRNAs) and microRNAs (miRNAs) in eukaryotes. RNase III proteins have been grouped in three major classes according to their domain organization. In this issue of Molecular Microbiology, Redko et al. identified a novel class of bacterial RNase III, named Mini-III, consisting only of the RNase III catalytic domain and functioning in the maturation of the 23S rRNA in Bacillus subtilis. Its absence from proteobacteria reveals that this step is mechanistically different from the corresponding step in Escherichia coli. The fact that Mini-III orthologues are present in unicellular photosynthetic eukaryotes and in plants opens new opportunities for functional studies of this type of RNases.

The lost world of Cuatro Ciénegas Basin, a relictual bacterial niche in a desert oasis.

Barriers to microbial migrations can lead adaptive radiations and increased endemism. We propose that extreme unbalanced nutrient stoichiometry of essential nutrients can be a barrier to microbial immigration over geological timescales. At the oasis in the Cuatro Ciénegas Basin in Mexico, nutrient stoichiometric proportions are skewed given the low phosphorus availability in the ecosystem. We show that this endangered oasis can be a model for a lost world. The ancient niche of extreme unbalanced nutrient stoichiometry favoured survival of ancestral microorganisms. This extreme nutrient imbalance persisted due to environmental stability and low extinction rates, generating a diverse and unique bacterial community. Several endemic clades of Bacillus invaded the Cuatro Cienegas region in two geological times, the late Precambrian and the Jurassic. Other lineages of Bacillus, Clostridium and Bacteroidetes migrated into the basin in isolated events. Cuatro Ciénegas Basin conservation is vital to the understanding of early evolutionary and ecological processes.

Individual variation in the advertisement call of Aplastodiscus albosignatus (Anura: Hylidae) is correlated with body size and environmental temperature

The importance of amphibian bioacoustics is widely documented in ecological, taxonomical and evolutionary studies, as calls might act as a primary mechanism of reproductive isolation. The influence of air temperature and male size on the variation of the advertisement calls in anurans is widely recognized but still insufficiently analyzed in Brazilian species. Our goals were to (1) describe the advertisement call of Aplastodiscus albosignatus (Lutz & Lutz, 1938), (2) test the influence of temperature and body size on its acoustic signals and (3) evaluate the variation of within and between-male acoustic signals. Advertisement calls of A. albosignatus consist of an unpulsed note with four harmonics. In most cases, the dominant frequency is the third harmonic but, in some calls, it was the minimum frequency (first harmonic). The average duration of calls was 0.191 s, the interval between calls was 2.08 s and the repetition rate was 33 calls per minute. On average, the minimum frequency was 550.15 Hz, the maximum frequency was 3531.70 Hz and the third harmonic was 2498.9 Hz. To evaluate the effect of air temperature, and body size on the variation of call parameters, we performed generalized linear models. The most explanatory model for spectral parameters was temperature plus body size. Concerning temporal variables, the best model that explains the variation in call duration was body size, while for the interval between calls was air temperature. The maximum frequency and the frequency of the third harmonic had little variation in the calls of both the same male and different males. Thus, these parameters were considered important in species recognition.(AU)

UVA Photoactivation of Harmol Enhances Its Antifungal Activity against the Phytopathogens Penicillium digitatum and Botrytis cinerea.

Phytopathogenic fungi responsible for post-harvest diseases on fruit and vegetables cause important economic losses. We have previously reported that harmol (1-methyl-9H-pyrido[3,4-b]indol-7-ol) is active against the causal agents of green and gray molds Penicillium digitatum and Botrytis cinerea, respectively. Here, antifungal activity of harmol was characterized in terms of pH dependency and conidial targets; also photodynamic effects of UVA irradiation on the antimicrobial action were evaluated. Harmol was able to inhibit the growth of both post-harvest fungal disease agents only in acidic conditions (pH 5), when it was found in its protonated form. Conidia treated with harmol exhibited membrane integrity loss, cell wall disruption, and cytoplasm disorganization. All these deleterious effects were more evident for B. cinerea in comparison to P. digitatum. When conidial suspensions were irradiated with UVA in the presence of harmol, antimicrobial activity against both pathogens was enhanced, compared to non-irradiated conditions. B. cinerea exhibited a high intracellular production of reactive oxygen species (ROS) when was incubated with harmol in irradiated and non-irradiated treatments. P. digitatum showed a significant increase in ROS accumulation only when treated with photoexcited harmol. The present work contributes to unravel the antifungal activity of harmol and its photoexcited counterpart against phytopathogenic conidia, focusing on ROS accumulation which could account for damage on different cellular targets.

Parallel Evolution and Horizontal Gene Transfer of the pst Operon in Firmicutes from Oligotrophic Environments.

The high affinity phosphate transport system (pst) is crucial for phosphate uptake in oligotrophic environments. Cuatro Cienegas Basin (CCB) has extremely low P levels and its endemic Bacillus are closely related to oligotrophic marine Firmicutes. Thus, we expected the pst operon of CCB to share the same evolutionary history and protein similarity to marine Firmicutes. Orthologs of the pst operon were searched in 55 genomes of Firmicutes and 13 outgroups. Phylogenetic reconstructions were performed for the pst operon and 14 concatenated housekeeping genes using maximum likelihood methods. Conserved domains and 3D structures of the phosphate-binding protein (PstS) were also analyzed. The pst operon of Firmicutes shows two highly divergent clades with no correlation to the type of habitat nor a phylogenetic congruence, suggesting horizontal gene transfer. Despite sequence divergence, the PstS protein had a similar 3D structure, which could be due to parallel evolution after horizontal gene transfer events.

Bacillus coahuilensis sp. nov., a moderately halophilic species from a desiccation lagoon in the Cuatro Ciénegas Valley in Coahuila, Mexico.

A moderately halophilic, Gram-positive and rod-shaped bacterium, strain m4-4T, was isolated from a Chihuahuan desert lagoon in Cuatro Ciénegas, Coahuila, Mexico. Strain m4-4T was found to grow optimally at 30-37 degrees C, pH 7.0-8.0 and 5 % NaCl and to tolerate from 0.5 % to 10 % NaCl. It was shown to be aerobic. The genomic DNA G+C content was about 37 mol%. Strain m4-4T exhibited minimal or no growth on most sugars tested. Its major cellular fatty acids were C14 : 0, C16 : 0 and C18 : 1. Based on phylogenetic analysis of 16S rRNA and recA gene sequences, we observed that the closest relatives of the isolate are moderately halophilic Bacillus species, with 16S rRNA gene sequence similarity ranging from 96.6 to 97.4 % (Bacillus marisflavi, Bacillus aquimaris and Bacillus vietnamensis). Additionally, using genomic data it was determined that the type strain contains a total of nine rRNA operons with three slightly different sequences. On the basis of phenotypic and molecular properties, strain m4-4T represents a novel species within the genus Bacillus, for which the name Bacillus coahuilensis sp. nov. is proposed, with the type strain m4-4T (=NRRL B-41737T =CECT 7197T).

ETHYLENE-INSENSITIVE5 encodes a 5'-->3' exoribonuclease required for regulation of the EIN3-targeting F-box proteins EBF1/2.

Ethylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence of ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F-box proteins, EBF1 and EBF2. Here we report the identification of ETHYLENE-INSENSITIVE5 as the 5'-->3' exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response.

Four distinct classes of proteins as interaction partners of the PABC domain of Arabidopsis thaliana Poly(A)-binding proteins.

Poly(A)-binding proteins (PABPs) play an important role in the regulation of translation and the control of mRNA stability in eukaryotes, and their functions are known to be essential in many organisms. PABPs contain a highly conserved C-terminal segment termed the PABC domain. The PABC domain from human PABP interacts with the proteins PAIP1, PAIP2 and RF3 via its PAM2 motifs. These interactions are important for modulating translation. Arabidopsis has eight PABPs, an unexpectedly large number in comparison to other eukaryotes whose genomes have been sequenced. Six of the Arabidopsis PABPs contain the conserved PABC domain. In this work, we have identified PABC-interacting proteins in Arabidopsis. Two proteins, which we named CID1 and CID7, were initially isolated in a two-hybrid screen, and eleven more were predicted to be present in the Arabidopsis proteome and eleven in the rice proteome. Among the 24 PAM2-containing proteins in this set, we observed a diversity of modules of intriguing function, ranging from acidic regions similar to the PAM1 motif found in human PAIP1 and PAIP2, to domains such as the small MutS-related domain, the Lsm domains of Ataxin-2, and RNA recognition motifs (RRMs). We suggest that the large number of PABPs and PAM2-containing proteins may have evolved to provide plants with greater flexibility in modulating the metabolism of specific transcripts. We also found that two PABP genes, PAB2 (ubiquitously expressed) and PAB5 (expressed in reproductive tissues), are essential for viability, suggesting that each has a vital and specific function.

Endospore degradation in an oligosporogenic, crystalliferous mutant of Bacillus thuringiensis.

We isolated a new oligosporogenic mutant from Bacillus thuringiensis var. kurstaki HD73 that retains the ability to produce insecticidal crystal inclusions. Sporulation in this mutant initiates in a manner similar to the wild-type strain, and under the electron microscope endospores are seen, but these do not reach maturity (except for 0.2% of them). At a late stage, the coat surrounding the forespore seems to lack shape and to be empty. Most mutant cells exhibit a well-formed bipyramidal crystal but are completely devoid of the forespore. The mutant has a functional SigK holoenzyme, which is required for the expression of genes involved in the formation of spore coat and cortex and for cry1A transcription from the BtII promoter. Defective maturation of spores could be due to an inadequate forespore coat or cortex structure resulting in the arrest of sporulation at late stage III or early stage IV.