Volatile organic compounds of the soil bacterium Bacillus halotolerans suppress pathogens and elicit defense-responsive genes in plants.

Volatile organic compounds (VOCs) produced by bacteria play an important, yet relatively unexplored role in interactions between plants and phytopathogens. In this study, the soil bacterium Bacillus halotolerans NYG5 was identified as a potent biocontrol agent against several phytopathogenic fungi (Macrophomina phaseolina, Rhizoctonia solani, Pythium aphanidermatum, and Sclerotinia sclerotiorum) through the production of VOCs. NYG5-emitted VOCs also inhibited the growth of bacterial pathogens (Agrobacterium tumefaciens, Xanthomonas campestris, Clavibacter michiganensis, and Pseudomonas syringae). When cultured in various growth media, NYG5 produced a variety of VOCs. Five distinct VOCs (2-methylbutanoic acid, 5-methyl-2-hexanone, 2,3-hexanedione, 2-ethyl-1-hexanol, and 6-methyl-2-heptanone) were identified using headspace GC-MS. 2,3-Hexanedione exhibited potent lethal effects on the tested phytopathogens and nematicidal activity against Meloidogyne javanica at a concentration of 50 ppm. In addition, 0.05 ppm 2,3-hexanedione stimulated the expression of pathogenesis-related genes 1 and 2 in Arabidopsis thaliana. Interestingly, 2,3-hexanedione is used as a food additive at higher concentrations than those tested in this study. Hence, 2,3-hexanedione is a promising biologically active compound that might serve as a sustainable alternative to common chemical pesticides and an elicitor of plant defense.

SlWRKY16 and SlWRKY31 of tomato, negative regulators of plant defense, involved in susceptibility activation following root-knot nematode Meloidogyne javanica infection.

The involvement of WRKY transcription factors in plant-nematode interactions, and in particular, how these WRKYs participate in regulating the complex morphological and physiological changes occurring after nematode infection, are the topic of active research. We characterized the functional role of the unstudied tomato WRKY genes SlWRKY16 and SlWRKY31 in regulating tomato roots' response to infection by the root-knot nematode Meloidogyne javanica. Using promoter-GUS reporter gene fusions and qRT-PCR, we show that both SlWRKYs are predominantly expressed during the first half of the parasitic life stages, when feeding-site induction and construction occur. Expression of SlWRKY16 increased sharply 15 days after inoculation, whereas SlWRKY31 was already induced earlier, but reached its maximum expression at this time. Both genes were downregulated at the mature female stage. To determine biological function, we produced transgenic lines overexpressing SlWRKY16 and SlWRKY31 in tomato hairy roots. Overexpression of both genes resulted in enhanced M. javanica infection, reflected by increased galling occurrence and reproduction. Expression profiling of marker genes responsive to defense-associated phytohormones indicated reductions in salicylic acid defense-related PR-1 and jasmonic acid defense-related PI in inoculated roots overexpressing SlWRK16 and SlWRKY31, respectively. Our results suggest that SlWRKY16 and SlWRKY31 function as negative regulators of plant immunity induced upon nematode infection.

Two Candidate Meloidogyne javanica Effector Genes, MjShKT and MjPUT3: A Functional Investigation of Their Roles in Regulating Nematode Parasitism.

During parasitism, root-knot nematode Meloidogyne spp. inject molecules termed effectors that have multifunctional roles in construction and maintenance of nematode feeding sites. As an outcome of transcriptomic analysis of Meloidogyne javanica, we identified and characterized two differentially expressed genes encoding the predicted proteins MjShKT, carrying a Stichodactyla toxin (ShKT) domain, and MjPUT3, carrying a ground-like domain, both expressed during nematode parasitism of the tomato plant. Fluorescence in-situ hybridization revealed expression of MjShKT and MjPUT3 in the dorsal esophageal glands, suggesting their injection into host cells. MjShKT expression was upregulated during the parasitic life stages, to a maximum at the mature female stage, whereas MjPUT3 expression increased in third- to fourth-stage juveniles. Subcellular in-planta localization of MjShKT and MjPUT3 using a fused fluorescence marker indicated MjShKT co-occurrence with the endoplasmic reticulum, the perinuclear endoplasmatic reticulum, and the Golgi organelle markers, while MjPUT3 localized, to some extent, within the endoplasmatic reticulum and was clearly observed within the nucleoplasm. MjShKT inhibited programmed cell death induced by overexpression of MAPKKKα and Gpa2/RBP-1. Overexpression of MjShKT in tomato hairy roots allowed an increase in nematode reproduction, as indicated by the high number of eggs produced on roots overexpressing MjShKT. Roots overexpressing MjPUT3 were characterized by enhanced root growth, with no effect on nematode development on those roots. Investigation of the two candidate effectors suggested that MjShKT is mainly involved in manipulating the plant effector-triggered immune response toward establishment and maintenance of active feeding sites, whereas MjPUT3 might modulate roots morphology in favor of nematode fitness in the host roots. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Minichromosome Maintenance Complex Component 2 (MjMCM2) of Meloidogyne javanica is a potential effector regulating the cell cycle in nematode-induced galls.

Root-knot nematodes Meloidogyne spp. induce enlarged multinucleate feeding cells-galls-in host plant roots. Although core cell-cycle components in galls follow a conserved track, they can also be usurped and manipulated by nematodes. We identified a candidate effector in Meloidogyne javanica that is directly involved in cell-cycle manipulation-Minichromosome Maintenance Complex Component 2 (MCM2), part of MCM complex licensing factor involved in DNA replication. MjMCM2, which is induced by plant oxilipin 9-HOT, was expressed in nematode esophageal glands, upregulated during parasitic stages, and was localized to plant cell nucleus and plasma membrane. Infected tomato hairy roots overexpressing MjMCM2 showed significantly more galls and egg-mass-producing females than wild-type roots, and feeding cells showed more nuclei. Phylogenetic analysis suggested seven homologues of MjMCM2 with unknown association to parasitism. Sequence mining revealed two RxLR-like motifs followed by SEED domains in all Meloidogyne spp. MCM2 protein sequences. The unique second RxLR-like motif was absent in other Tylenchida species. Molecular homology modeling of MjMCM2 suggested that second RxLR2-like domain is positioned on a surface loop structure, supporting its function in polar interactions. Our findings reveal a first candidate cell-cycle gene effector in M. javanica-MjMCM2-that is likely secreted into plant host to mimic function of endogenous MCM2.

Tomato Divinyl Ether-Biosynthesis Pathway Is Implicated in Modulating of Root-Knot Nematode Meloidogyne javanica's Parasitic Ability.

The role of the 9-lipoxygenase (9-LOX)-derived oxylipins in plant defense is mainly known in solanaceous plants. In this work, we identify the functional role of the tomato divinyl ether synthase (LeDES) branch, which exclusively converts 9-hydroperoxides to the 9-divinyl ethers (DVEs) colneleic acid (CA) and colnelenic acid (CnA), during infection by the root-knot nematode Meloidogyne javanica. Analysis of LeDES expression in roots indicated a concurrent response to nematode infection, demonstrating a sharp increase in expression during the molting of third/fourth-stage juveniles, 15 days after inoculation. Spatiotemporal expression analysis using an LeDES promoter:GUS tomato line showed high GUS activity associated with the developing gall; however the GUS signal became more constricted as infection progressed to the mature nematode feeding sites, and eventually disappeared. Wounding did not activate the LeDES promoter, but auxins and methyl salicylate triggered LeDES expression, indicating a hormone-mediated function of DVEs. Heterologous expression of LeDES in Arabidopsis thaliana rendered the plants more resistant to nematode infection and resulted in a significant reduction in third/fourth-stage juveniles and adult females as compared to a vector control and the wild type. To further evaluate the nematotoxic activity of the DVEs CA and CnA, recombinant yeast that catalyzes the formation of CA and CnA from 9-hydroperoxides was generated. Transgenic yeast accumulating CnA was tested for its impact on M. javanica juveniles, indicating a decrease in second-stage juvenile motility. Taken together, our results suggest an important role for LeDES as a determinant in the defense response during M. javanica parasitism, and indicate two functional modes: directly via DVE motility inhibition effect and through signal molecule-mediated defense reactions to nematodes that depend on methyl salicylate.

Oxylipins are implicated as communication signals in tomato-root-knot nematode (Meloidogyne javanica) interaction.

Throughout infection, plant-parasitic nematodes activate a complex host defense response that will regulate their development and aggressiveness. Oxylipins-lipophilic signaling molecules-are part of this complex, performing a fundamental role in regulating plant development and immunity. At the same time, the sedentary root-knot nematode Meloidogyne spp. secretes numerous effectors that play key roles during invasion and migration, supporting construction and maintenance of nematodes' feeding sites. Herein, comprehensive oxylipin profiling of tomato roots, performed using LC-MS/MS, indicated strong and early responses of many oxylipins following root-knot nematode infection. To identify genes that might respond to the lipidomic defense pathway mediated through oxylipins, RNA-Seq was performed by exposing Meloidogyne javanica second-stage juveniles to tomato protoplasts and the oxylipin 9-HOT, one of the early-induced oxylipins in tomato roots upon nematode infection. A total of 7512 differentially expressed genes were identified. To target putative effectors, we sought differentially expressed genes carrying a predicted secretion signal peptide. Among these, several were homologous with known effectors in other nematode species; other unknown, potentially secreted proteins may have a role as root-knot nematode effectors that are induced by plant lipid signals. These include effectors associated with distortion of the plant immune response or manipulating signal transduction mediated by lipid signals. Other effectors are implicated in cell wall degradation or ROS detoxification at the plant-nematode interface. Being an integral part of the plant's defense response, oxylipins might be placed as important signaling molecules underlying nematode parasitism.

The Genus Pratylenchus (Nematoda: Pratylenchidae) in Israel: From Taxonomy to Control Practices.

Due to Israel's successful agricultural production and diverse climatic conditions, plant-parasitic nematodes are flourishing. The occurrence of new, previously unidentified species in Israel or of suggested new species worldwide is a consequence of the continuous withdrawal of efficient nematicides. Among plant-parasitic nematodes, migratory endoparasitic species of the genus Pratylenchus are widely distributed in vegetable and crop fields in Israel and are associated with major reductions in quality and yield. This review focuses on the occurrence, distribution, diagnosis, pathogenicity, and phylogeny of all Pratylenchus species recorded over the last few decades on different crops grown throughout Israel-covering early information from nematologists to recent reports involving the use of molecular phylogenetic methodologies. We explore the accepted distinction between Pratylenchus thornei and Pratylenchus mediterraneus isolated from Israel's northern Negev region, and address the confusion concerning the findings related to these Pratylenchus species. Our recent sampling from the northern Negev revealed the occurrence of both P. thornei and P. mediterraneus on the basis of molecular identification, indicating P. mediterraneus as a sister species of P. thornei and their potential occurrence in a mixed infection. Finally, the efficiencies of common control measures taken to reduce Pratylenchus' devastating damage in protected crops and field crops is discussed.

Real-Time Visualization of Cellulase Activity by Microorganisms on Surface.

A variety of methods to detect cellulase secretion by microorganisms has been developed over the years, none of which enables the real-time visualization of cellulase activity on a surface. This visualization is critical to study the interaction between soil-borne cellulase-secreting microorganisms and the surface of plant roots and specifically, the effect of surface features on this interaction. Here, we modified the known carboxymethyl cellulase (CMC) hydrolysis visualization method to enable the real-time tracking of cellulase activity of microorganisms on a surface. A surface was formed using pure CMC with acridine orange dye incorporated in it. The dye disassociated from the film when hydrolysis occurred, forming a halo surrounding the point of hydrolysis. This enabled real-time visualization, since the common need for post hydrolysis dyeing was negated. Using root-knot nematode (RKN) as a model organism that penetrates plant roots, we showed that it was possible to follow microorganism cellulase secretion on the surface. Furthermore, the addition of natural additives was also shown to be an option and resulted in an increased RKN response. This method will be implemented in the future, investigating different microorganisms on a root surface microstructure replica, which can open a new avenue of research in the field of plant root-microorganism interactions.

PPNID: a reference database and molecular identification pipeline for plant-parasitic nematodes.

MOTIVATION: The phylum Nematoda comprises the most cosmopolitan and abundant metazoans on Earth and plant-parasitic nematodes represent one of the most significant nematode groups, causing severe losses in agriculture. Practically, the demands for accurate nematode identification are high for ecological, agricultural, taxonomic and phylogenetic researches. Despite their importance, the morphological diagnosis is often a difficult task due to phenotypic plasticity and the absence of clear diagnostic characters while molecular identification is very difficult due to the problematic database and complex genetic background. RESULTS: The present study attempts to make up for currently available databases by creating a manually-curated database including all up-to-date authentic barcoding sequences. To facilitate the laborious process associated with the interpretation and identification of a given query sequence, we developed an automatic software pipeline for rapid species identification. The incorporated alignment function facilitates the examination of mutation distribution and therefore also reveals nucleotide autapomorphies, which are important in species delimitation. The implementation of genetic distance, plot and maximum likelihood phylogeny analysis provides more powerful optimality criteria than similarity searching and facilitates species delimitation using evolutionary or phylogeny species concepts. The pipeline streamlines several functions to facilitate more precise data analyses, and the subsequent interpretation is easy and straightforward. AVAILABILITY AND IMPLEMENTATION: The pipeline was written in vb.net, developed on Microsoft Visual Studio 2017 and designed to work in any Windows environment. The PPNID is distributed under the GNU General Public License (GPL). The executable file along with tutorials is available at https://github.com/xueqing4083/PPNID. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Tomato SlWRKY3 acts as a positive regulator for resistance against the root-knot nematode Meloidogyne javanica by activating lipids and hormone-mediated defense-signaling pathways.

Diseases caused by plant-parasitic nematodes in vegetables, among them Meloidogyne spp. root-knot nematodes (RKNs), lead to extensive yield decline. A molecular understanding of the mechanisms underlying plants' innate resistance may enable developing safe alternatives to harmful chemical nematicides in controlling RKNs. A tight relationship has been revealed between the WRKY transcription factors and RKN parasitism on tomato roots. We investigated the function role of tomato SlWRK3 and SlWRKY35 in regulating nematode disease development. Using promoter-GUS reporter gene fusions, we show that both SlWRKY3 and SlWRKY35 are induced within 5 days of infection and through feeding-site development and gall maturation, with a much stronger response of the former vs. the latter to nematode infection. Histological analysis of nematode-feeding sites indicated a high expression of SlWRKY3 in developing and mature feeding cells and associated vasculature cells, whereas SlWRKY35 expression was only observed in mature feeding sites. Both SlWRKY3 and SlWRKY35 promoters were induced by the defense phytohormones salicylic acid and indole-3-butyric acid, with no response to either jasmonic acid or methyl jasmonate. SlWRKY3 overexpression resulted in lower infection of the RKN Meloidogyne javanica, whereas knocking down SlWRKY3 resulted in increased infection. Phytohormone and oxylipin profiles determined by LC-MS/MS showed that the enhanced resistance in the former is coupled with an increased accumulation of defense molecules from the shikimate and oxylipin pathways. Our results pinpoint SlWRKY3 as a positive regulator of induced resistance in response to nematode invasion and infection, mostly during the early stages of nematode infection.

Phylogeography and Molecular Species Delimitation of Pratylenchus capsici n. sp., a New Root-Lesion Nematode in Israel on Pepper (Capsicum annuum).

Root-lesion nematodes of the genus Pratylenchus parasitize the roots of numerous plants and can cause severe damage and yield loss. Here, we report on a new species, Pratylenchus capsici n. sp., from the Arava rift, Israel, which was characterized by integrative methods, including detailed morphology, molecular phylogeny, population genetics, and phylogeography. This species is widely spread across the Arava rift, causing significant infection in pepper (Capsicum annuum) roots and inhibiting plant growth. Both morphological and molecular species delimitation support the recovered species as a new species. We found high cytochrome oxidase subunit I haplotype diversity, and phylogeography analysis suggests that contemporary gene flow is prevented among different agricultural farms, while population dispersal from weeds (Chenopodium album and Sonchus oleraceus) to pepper occurs on a relatively small scale. Our results suggest that weeds are an important reservoir for the dispersal of P. capsici n. sp., either as the original nematode source or at least in maintaining the population between growing seasons.

Caenorhabditis elegans susceptibility to Daldinia cf. concentrica bioactive volatiles is coupled with expression activation of the stress-response transcription factor daf-16, a part of distinct nematicidal action.

The bionematicidal effect of a synthetic volatile mixture (SVM) of four volatile organic compounds (VOCs) emitted by the endophytic fungus Daldinia cf. concentrica against the devastating plant-parasitic root-knot nematode Meloidogyne javanica has been recently demonstrated in both in vitro and greenhouse experiments. However, the mode of action governing the observed irreversible paralysis of J2 larvae upon exposure to SVM is unknown. To unravel the mechanism underlying the anthelmintic and nematicidal activities, we used the tractable model worm Caenorhabditis elegans. C. elegans was also susceptible to both the fungal VOCs and SVM. Among compounds comprising SVM, 3-methyl-1-butanol, (±)-2-methyl-1-butanol, and 4-heptanone showed significant nematicidal activity toward L1, L4 and young adult stages. Egg hatching was only negatively affected by 4-heptanone. To determine the mechanism underlying this activity, we examined the response of C. elegans mutants for glutamate-gated chloride channel and acetylcholine transporter, targets of the nematicidal drugs ivermectin and aldicarb, respectively, to 4-heptanone and SVM. These aldicarb- and ivermectin-resistant mutants retained susceptibility upon exposure to 4-heptanone and SVM. Next, we used C. elegans TJ356 strain zIs356 (daf-16::GFP+rol-6), LD1 ldIs7 [skn-1B/C::GFP + pRF4(rol-6(su1006))], LD1171 ldIs3 [gcs-1p::gfp; rol-6(su1006))], CL2166 dvIs19 (gst-4p::GFP) and CF1553 muIs84 (sod-3p::GFP+rol-6), which have mutations in genes regulating multiple stress responses. Following exposure of L4 larvae to 4-heptanone or SVM, there was clear nuclear translocation of DAF-16::GFP, and SKN-1::GFP indicating that their susceptibility involves DAF-16 and SKN1 regulation. Application of 4-heptanone, but not SVM, induced increased expression of, gcs-1::GFP and gst-4::GFP compared to controls. In contrast, application of 4-heptanone or SVM to the sod-3::GFP line elicited a significant decline in overall fluorescence intensity compared to controls, indicating SOD-3 downregulation and therefore overall reduction in cellular redox machinery. Our data indicate that the mode of action of SVM and 4-heptanone from D. cf. concentrica differs from that of currently available nematicides, potentially offering new solutions for nematode management.

SlWRKY45, nematode-responsive tomato WRKY gene, enhances susceptibility to the root knot nematode; M. javanica infection.

The fluctuation of tomato's WRKY defense regulators during infection by the root knot nematode Meloidogyne javanica was analyzed: and the spatial and temporal expression of SlWRKY45 was studied in depth with regard to its response to nematode infection, phytohormones, and wounding. Expression of WRKY45 increased substantially within 5 d upon infection and continued through feeding-site development and gall maturation. Histological analysis of nematode feeding sites indicated that WRKY45 was highly expressed within the feeding cells and associated vascular parenchyma cells. Responses of SlWRKY45 promoters to several phytohormones showed that WRKY45 was highly induced by specific phytohormones, including cytokinin, auxin, and the defense-signaling molecule salicylic acid (SA), but not by the jasmonates. Overexpressing tomato lines were generated, and infection tests showed that, significantly, roots over-expressing SlWRKY45 contained substantially increased number of females, indicating that WRKY45 overexpression supported faster nematode development. qRT-PCR tests have shown roots overexpressing WRKY45 suppressed the jasmonic acid and salicylic acid marker genes, proteinase inhibitor (PI), and pathogenesis related protein (PR1), respectively, and also the cytokinin response factors CRF1 and CRF6. Overall, this study indicated SlWRKY45 to be a potential transcription factor whose manipulation by the invading nematode might be critical for coordination of hormone signals supporting favorable condition for nematode development in root tissue.

Pathogenic Variability of Meloidogyne incognita Populations Occurring in Pepper-Production Greenhouses in Israel Toward Me1, Me3 and N Pepper Resistance Genes.

Natural variation in the root-knot nematode Meloidogyne incognita is problematic for breeding programs: populations possessing similar morphological characteristics can produce different reactions on the same host. We collected 30 widely dispersed M. incognita populations from protected pepper production systems in major pepper-growing regions of Israel and accurately identified their virulence characteristics by modified differential host test in a growth chamber on tomato, tobacco, cotton, melon, pepper, and peanut. Galling indices and reproduction were determined on the different hosts. All populations fit the published scheme for M. incognita race 2, except for reproduction on cotton plants by five out of 25 tested M. incognita populations, indicating host-range variations. Reaction of three genes that confer resistance to M. incognita-Me1, Me3 and N-to the collected populations was evaluated. Several M. incognita populations induced galling and reproduced successfully on pepper genotypes carrying Me3 and N, whereas plant resistance conferred by Me1 was more robust for all examined populations. Moreover, the effect of genetic background on Me1 resistance demonstrated a relative advantage of several genotypes in nematode infestations. Efficiency of Me3 under local nematode infestation was further studied with a homozygous line carrying two Me3 alleles. Reproduction of virulent populations on the homozygotes (Me3/Me3) and heterozygotes (Me3/Me3+) was similar, suggesting a limited quantitative effect of Me3. These results present the first characterization of host range, reproduction, and molecular aspects of M. incognita from Israel and highlight the importance of taking a multidimensional approach in pepper-breeding programs for resistance to M. incognita.

Bioactive Volatiles from an Endophytic Daldinia cf. concentrica Isolate Affect the Viability of the Plant Parasitic Nematode Meloidogyne javanica.

Plant-parasitic nematodes form one of the largest sources of biotic stress imposed on plants, and are very difficult to control; among them are the obligate parasites, the sedentary root-knot nematodes (RKNs)-Meloidogyne spp.-which are extremely polyphagous and exploit a very wide range of hosts. Endophytic fungi are organisms that spend most of their life cycle within plant tissue without causing visible damage to the host plant. Many endophytes secrete specialized metabolites and/or emit volatile organic compounds (VOCs) that exhibit biological activity. Recently, we demonstrated that the endophytic fungus Daldinia cf. concentrica secrets biologically active VOCs. Here we examined the ability of the fungus and its VOCs to control the RKN M. javanica both in vitro and greenhouse experiments. The D. cf. concentrica VOCs showed bionematicidal activity against the second-stage juveniles (J2s) of M. javanica. We found that exposure of J2s to fungal volatiles caused 67% reduction in viability, and that application of a synthetic volatile mixture (SVM), comprising 3-methyl-1-butanol, (±)-2-methyl-1-butanol, 4-heptanone, and isoamyl acetate, in volumetric ratio of 1:1:2:1 further reduced J2s viability by 99%. We demonstrated that, although each of the four VOCs significantly reduced the viability of J2s relative to the control, only 4-heptanone elicited the same effect as the whole mixture, with nematicidal activity of 90% reduction in viability of the J2s. Study of the effect of the SVM on egg hatching demonstrated that it decreased eggs hatching by 87%. Finally, application of the SVM to soil inoculated with M. javanica eggs or J2s prior to planting susceptible tomato plants resulted in a significantly reduced galling index and fewer eggs produced on each root system, with no effect on root weight. Thus, D. cf. concentrica and/or SVM based on fungal VOCs may be considered as a novel alternative approach to controlling the RKN M. javanica.

A salinity and sulfate manipulation of hypersaline microbial mats reveals stasis in the cyanobacterial community structure.

The cyanobacterial community structure and composition of hypersaline mats were characterized in an experiment in which native salinity and sulfate levels were modified. Over the course of approximately 1 year, microbial mats collected from Guerrero Negro (Baja, California Sur, Mexico) were equilibrated to lowered salinity (to 35 p.p.t.) and lowered sulfate (below 1 mM) conditions. The structure and composition of the cyanobacterial community in the top 5 mm of these mats were examined using a multifaceted cultivation-independent molecular approach. Overall, the relative abundance of cyanobacteria-roughly 20% of the total bacterial community, as assayed with a PCR-based methodology-was not significantly affected by these manipulations. Furthermore, the mat cyanobacterial community was only modestly influenced by the dramatic changes in sulfate and salinity, and the dominant cyanobacteria were unaffected. Community composition analyses confirmed the dominant presence of the cosmopolitan cyanobacterium Microcoleus chthonoplastes, but also revealed the dominance of another Oscillatorian cyanobacterial group, also detected in other hypersaline microbial mats. Cyanobacterial populations increasing in relative abundance under the modified salinity and sulfate conditions were found to be most closely related to other hypersaline microbial mat organisms, suggesting that the development of these mats under native conditions precludes the development of organisms better suited to the less restrictive experimental conditions. These results also indicate that within a significant range of salinity and sulfate concentrations, the cyanobacterial community is remarkably stable.