A subclass II bHLH transcription factor in Marchantia polymorpha gives insight into the ancestral land plant trait of spore formation.

Sporopollenin is often said to be one of the toughest biopolymers known to man. The shift in dormancy cell wall deposition from around the diploid zygotes of charophycean algae to sporopollenin around the haploid spores of land plants essentially imparted onto land plants the gift of passive motility, a key acquisition that contributed to their vast and successful colonization across terrestrial habitats.1,2 A putative transcription factor controlling the land plant mode of sporopollenin deposition is the subclass II bHLHs, which are conserved and novel to land plants, with mutants of genes in angiosperms and mosses divulging roles relating to tapetum degeneration and spore development.3,4,5,6,7 We demonstrate that a subclass II bHLH gene, MpbHLH37, regulates sporopollenin biosynthesis and deposition in the model liverwort Marchantia polymorpha. Mpbhlh37 sporophytes show a striking loss of secondary wall deposits of the capsule wall, the elaters, and the spore exine, all while maintaining spore viability, identifying MpbHLH37 as a master regulator of secondary wall deposits of the sporophyte. Localization of MpbHLH37 to the capsule wall and elaters of the sporophyte directly designates these tissue types as a bona fide tapetum in liverworts, giving support to the notion that the presence of a tapetum is an ancestral land plant trait. Finally, as early land plant spore walls exhibit evidence of tapetal deposition,8,9,10,11,12 a tapetal capsule wall could have provided these plants with a developmental mechanism for sporopollenin deposition.

Genomes tell tales of spores versus seeds.

Genomes from ferns and a cycad reveal deep roots of plant reproduction.

Plastid Transformation of Sporelings from the Liverwort Marchantia polymorpha L.

We describe a simple and efficient plastid transformation method for the liverwort, Marchantia polymorpha L. Use of rapidly proliferating cells such as sporelings, which are immature thalli developing from spores, as targets made plastid transformation by particle bombardment efficient. Selection on a sucrose-free medium and linearization of the transformation vector significantly improved the recovery rate of plastid transformants. With the method described here, a few plastid transformants are obtained from a single bombardment of sporelings. Homoplasmic transformants of thalli are obtained immediately after primary selection.

Ubiquitin-dependent Argonauteprotein MEL1 degradation is essential for rice sporogenesis and phasiRNA target regulation.

MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice (Oryza sativa) Argonaute (AGO) protein, has been reported to function specifically at premeiotic and meiotic stages of germ cell development and is associated with a novel class of germ cell-specific small noncoding RNAs called phased small RNAs (phasiRNAs). MEL1 accumulation is temporally and spatially regulated and is eliminated after meiosis. However, the metabolism and turnover (i.e. the homeostasis) of MEL1 during germ cell development remains unknown. Here, we show that MEL1 is ubiquitinated and subsequently degraded via the proteasome pathway in vivo during late sporogenesis. Abnormal accumulation of MEL1 after meiosis leads to a semi-sterile phenotype. We identified a monocot-specific E3 ligase, XBOS36, a CULLIN RING-box protein, that is responsible for the degradation of MEL1. Ubiquitination at four K residues at the N terminus of MEL1 by XBOS36 induces its degradation. Importantly, inhibition of MEL1 degradation either by XBOS36 knockdown or by MEL1 overexpression prevents the formation of pollen at the microspore stage. Further mechanistic analysis showed that disrupting MEL1 homeostasis in germ cells leads to off-target cleavage of phasiRNA target genes. Our findings thus provide insight into the communication between a monocot-specific E3 ligase and an AGO protein during plant reproductive development.

Polyploid progeny from triploid hybrids of Phegopteris decursivepinnata (Thelypteridaceae).

Phegopteris decursivepinnata includes diploids, tetraploids, and triploid hybrids based on x = 30. We obtained polyploid progeny from triploid hybrids through selfing and crossing experiments. Triploids occasionally formed well-filled spores. The mean occurrence frequencies of well-filled and germinated spores were 2.8% and 0.8%, respectively. Viable spores that succeeded in germinating were regarded as unreduced, triploid spores, because the resulting gametophytes yielded triploid (2n = 86-92) and hexaploid (2n = 170-184) progeny in both isolated and mixed cultures of gametophytes. The triploid and hexaploid progeny likely arose apogamously and sexually, respectively. One of the hexaploid progeny yielded hexaploid sporophytes (2n = 169-180) in the mixed culture of its gametophytes. Artificial crossing between triploid and diploid sporophytes produced tetraploid (2n = 116, 120) and pentaploid (2n = 145-150) progeny that likely arose through the mating of 3x gametes from the triploid with both 1x and 2x gametes from the diploid, respectively. Unreduced spore formation was confirmed in diploid sporophytes. The tetraploid progeny formed viable spores at a frequency of 63-75%. Triploid hybrids of this species are thus expected to produce new triploids, tetraploids, and hexaploids in nature. The wide range of variation in chromosome numbers of hexaploid progeny suggests that viable spores from parental triploid hybrids had unreduced chromosomes, whose numbers, however, deviated considerably from those of the hybrids. This chromosome deviation of viable spores may result from errant movements of chromatids of univalents when unreduced dyads form in meiosis. Downward chromosome deviation from the chromosome number of the parental hybrids may affect the developmental progress of viable spores more tolerantly than upward chromosome deviation.

Phytopathogenic oomycetes: a review focusing on Phytophthora cinnamomi and biotechnological approaches.

The Phytophthora genus is composed, mainly, of plant pathogens. This genus belongs to the Oomycete class, also known as "pseudo-fungi", within the Chromista Kingdom. Phytophthora spp. is highlighted due to the significant plant diseases that they cause, which represents some of the most economically and cultural losses, such as European chestnut ink disease, which is caused by P. cinnamomi. Currently, there have been four genome assemblies placed at the National Center for Biotechnology Information (NCBI), although the progress to understand and elucidate the pathogenic process of P. cinnamomi by its genome is progressing slowly. In this review paper, we aim to report and discuss the recent findings related to P. cinnamomi and its genomic information. Our research is based on paper databases that reported probable functions to P. cinnamomi proteins using sequence alignments, bioinformatics, and biotechnology approaches. Some of these proteins studied have functions that are proposed to be involved in the asexual sporulation and zoosporogenesis leading to the host colonization and consequently associated with pathogenicity. Some remarkable genes and proteins discussed here are related to oospore development, inhibition of sporangium formation and cleavage, inhibition of flagellar assembly, blockage of cyst germination and hyphal extension, and biofilm proteins. Lastly, we report some biotechnological approaches using biological control, studies with genome sequencing of P. cinnamomi resistant plants, and gene silencing through RNA interference (iRNA).

Barcoded microbial system for high-resolution object provenance.

Determining where an object has been is a fundamental challenge for human health, commerce, and food safety. Location-specific microbes in principle offer a cheap and sensitive way to determine object provenance. We created a synthetic, scalable microbial spore system that identifies object provenance in under 1 hour at meter-scale resolution and near single-spore sensitivity and can be safely introduced into and recovered from the environment. This system solves the key challenges in object provenance: persistence in the environment, scalability, rapid and facile decoding, and biocontainment. Our system is compatible with SHERLOCK, a Cas13a RNA-guided nucleic acid detection assay, facilitating its implementation in a wide range of applications.

Evidence of defined temporal expression patterns that lead a gram-negative cell out of dormancy.

Many bacterial species are capable of forming long-lived dormant cells. The best characterized are heat and desiccation resistant spores produced by many Gram-positive species. Less characterized are dormant cysts produced by several Gram-negative species that are somewhat tolerant to increased temperature and very resistant to desiccation. While there is progress in understanding regulatory circuits that control spore germination, there is scarce information on how Gram-negative organisms emerges from dormancy. In this study, we show that R. centenum cysts germinate by emerging a pair of motile vegetative cells from a thick cyst cell wall coat ~ 6 hrs post induction of germination. Time-lapse transcriptomic analysis reveals that there is a defined temporal pattern of gene expression changes during R. centenum cyst germination. The first observable changes are increases in expression of genes for protein synthesis, an increase in expression of genes involved in the generation of a membrane potential and the use of this potential for ATP synthesis via ATPase expression. These early events are followed by expression changes that affect the cell wall and membrane composition, followed by expression changes that promote chromosome replication. Midway through germination, expression changes occur that promote the flow of carbon through the TCA cycle to generate reducing power and parallel synthesis of electron transfer components involved in oxidative phosphorylation. Finally, late expression changes promote the synthesis of a photosystem as well as flagellar and chemotaxis components for motility.

Dynamic Extreme Aneuploidy (DEA) in the vegetable pathogen Phytophthora capsici and the potential for rapid asexual evolution.

Oomycete plant pathogens are difficult to control and routine genetic research is challenging. A major problem is instability of isolates. Here we characterize >600 field and single zoospore isolates of Phytophthora capsici for inheritance of mating type, sensitivity to mefenoxam, chromosome copy number and heterozygous allele frequencies. The A2 mating type was highly unstable with 26% of 241 A2 isolates remaining A2. The A1 mating type was stable. Isolates intermediately resistant to mefenoxam produced fully resistant single-spore progeny. Sensitive isolates remained fully sensitive. Genome re-sequencing of single zoospore isolates revealed extreme aneuploidy; a phenomenon dubbed Dynamic Extreme Aneuploidy (DEA). DEA is characterized by the asexual inheritance of diverse intra-genomic combinations of chromosomal ploidy ranging from 2N to 3N and heterozygous allele frequencies that do not strictly correspond to ploidy. Isolates sectoring on agar media showed dramatically altered heterozygous allele frequencies. DEA can explain the rapid increase of advantageous alleles (e.g. drug resistance), mating type switches and copy neutral loss of heterozygosity (LOH). Although the mechanisms driving DEA are unknown, it can play an important role in adaptation and evolution and seriously hinders all aspects of P. capsici research.

G protein α subunit suppresses sporangium formation through a serine/threonine protein kinase in Phytophthora sojae.

Eukaryotic heterotrimeric guanine nucleotide-binding proteins consist of α, ß, and γ subunits, which act as molecular switches to regulate a number of fundamental cellular processes. In the oomycete pathogen Phytophthora sojae, the sole G protein α subunit (Gα; encoded by PsGPA1) has been found to be involved in zoospore mobility and virulence, but how it functions remains unclear. In this study, we show that the Gα subunit PsGPA1 directly interacts with PsYPK1, a serine/threonine protein kinase that consists of an N-terminal region with unknown function and a C-terminal region with a conserved catalytic kinase domain. We generated knockout and knockout-complemented strains of PsYPK1 and found that deletion of PsYPK1 resulted in a pronounced reduction in the production of sporangia and oospores, in mycelial growth on nutrient poor medium, and in virulence. PsYPK1 exhibits a cytoplasmic-nuclear localization pattern that is essential for sporangium formation and virulence of P. sojae. Interestingly, PsGPA1 overexpression was found to prevent nuclear localization of PsYPK1 by exclusively binding to the N-terminal region of PsYPK1, therefore accounting for its negative role in sporangium formation. Our data demonstrate that PsGPA1 negatively regulates sporangium formation by repressing the nuclear localization of its downstream kinase PsYPK1.

Henneguya (Cnidaria: Myxosporea: Myxobolidae) infections of cultured barramundi, Lates calcarifer (Perciformes: Latidae) in an estuarine wetlands system of Malaysia: description of Henneguya setiuensis n. sp., Henneguya voronini n. sp. and Henneguya calcarifer n. sp.

Examination of 35 barramundi (Lates calcarifer) from aquaculture cages in Setiu Wetland, Malaysia, revealed a single fish infected with three Henneguya spp. (Cnidaria: Myxosporea). Characterization of the infections using tissue tropism, myxospore morphology and morphometry and 18S rDNA sequencing supported description of three new species: Henneguya setiuensis n. sp., Henneguya voronini n. sp. and H. calcarifer n. sp. Myxospores of all three species had typical Henneguya morphology, with two polar capsules in the plane of the suture, an oval spore body, smooth valve cell surfaces, and two caudal appendages. Spores were morphometrically similar, and many dimensions overlapped, but H. voronini n. sp. had shorter caudal appendages compared with H. calcarifer n. sp. and H. setiuensis n. sp. Gross tissue tropism distinguished the muscle parasite H. calcarifer n. sp. from gill parasites H. setiuensis n. sp. and H. voronini n. sp.; and these latter two species were further separable by fine-scale location of developing plasmodia, which were intra-lamellar for H. setiuensis n. sp. and basal to the filaments for H. voronini n. sp. small subunit ribosomal DNA sequences distinguished all three species: the two gill species H. setiuensis n. sp. and H voronini n. sp. were only 88% similar (over 1708 bp), whereas the muscle species H. calcarifer n. sp. was most similar to H. voronini n. sp. (98% over 1696 bp). None of the three novel species was more than 90% similar to any known myxosporean sequence in GenBank. Low infection prevalence of these myxosporeans and lack of obvious tissue pathology from developing plasmodia suggested none of these parasites are currently a problem for barramundi culture in Setiu Wetland; however additional surveys of fish, particularly at different times of the year, would be informative for better risk assessment.

Cyclic AMP induction of Dictyostelium prespore gene expression requires autophagy.

Dictyostelium discoideum amoebas display colonial multicellularity where starving amoebas aggregate to form migrating slugs and fruiting bodies consisting of spores and three supporting cell types. To resolve the cell signalling mechanism that control sporulation, we use insertional mutagenesis of amoebas transformed with fusion constructs of spore genes and red fluorescent protein. We identified the defective gene in a mutant lacking spore gene expression as the autophagy gene Atg7. Directed knock-out of atg7 and of autophagy genes like atg5 and atg9 yielded a similar phenotype, with lack of viable spores and excessive differentiation of stalk cells. The atg7-, atg5- and atg9- cells were specifically defective in cAMP induction of prespore genes, but showed enhanced cAMP stimulation of prestalk genes at the same developmental stage. The lack of prespore gene induction in the autophagy mutants was not due to deleterious effects of loss of autophagy on known components of the cAMP pathway, such as cAMP receptors and their cAMP-induced phosphorylation and internalization, PKA and the transcription factors SpaA and GbfA, or to lack of NH3 production by proteolysis, which was previously suggested to stimulate the spore pathway. Our continued mutagenesis approach is the most likely to yield the intriguing link between autophagy and prespore gene induction.

Morphological and molecular characterization of Ceratomyxa batam n. sp. (Myxozoa: Ceratomyxidae) infecting the gallbladder of the cultured Trachinotus ovatus (Perciformes: Carangidae) in Batam Island, Indonesia.

A new coelozoic myxozoan species, Ceratomyxa batam n. sp., was identified in cultured carangid fish, Trachinotus ovatus (Perciformes: Carangidae), in waters off Batam Island of Indonesia. The bi- and trivalved spores were observed in the gallbladder of T. ovatus. Mature bivalved spores of C. batam n. sp. were transversely elongated and narrowly crescent in shape, 3.8 ± 0.36 (2.7-4.6) µm long and 19.2 ± 1.75 (16.2-22.0) µm thick. Two sub-spherical polar capsules were 2.3 ± 0.18 (2.0-2.8) µm long and 2.6 ± 0.16 (2.3-2.9) µm wide. Prevalence was 72.2% in 72 examined T. ovatus according to evaluations dating from November 2016. The maximum likelihood phylogenetic tree based on small subunit rDNA sequence showed similarity with Ceratomyxa robertsthomsoni and Ceratomyxa thalassomae found in Australia. This is the first report of Ceratomyxa species identified in a seawater fish at Batam Island, Indonesia.

Psychedelic fungus (Psilocybe sp.) authentication in a case of illegal drug traffic: sporological, molecular analysis and identification of the psychoactive substance.

In nature, there are >200 species of fungi with hallucinogenic properties. These fungi are classified as Psilocybe, Gymnopilus, and Panaeolus which contain active principles with hallucinogenic properties such as ibotenic acid, psilocybin, psilocin, or baeocystin. In Chile, fungi seizures are mainly of mature specimens or spores. However, clandestine laboratories have been found that process fungus samples at the mycelium stage. In this transient stage of growth (mycelium), traditional taxonomic identification is not feasible, making it necessary to develop a new method of study. Currently, DNA analysis is the only reliable method that can be used as an identification tool for the purposes of supporting evidence, due to the high variability of DNA between species. One way to identify the species of a distinctive DNA fragment is to study PCR products analyzed by real time PCR and sequencing. One of the most popular sequencing methods of forensic interest at the generic and intra-generic levels in plants is internal transcribed spacer (ITS). With real time PCR it is possible to distinguish PCR products by differential analysis of their melting temperature (Tm) curves. This paper describes morphological, chemical, and genetic analysis of mycelia of psychedelic fungi collected from a clandestine laboratory. The fungus species were identified using scanning electron microscopy (SEM), mass spectrometry, HRM analysis, and ITS sequencing. The sporological studies showed a generally smooth surface and oval shape, with maximum length 10.1 µm and width 6.4 µm. The alkaloid Psilocyn was identified by mass spectrometry, while HRM analysis and ITS sequencing identified the species as Psilocybe cubensis. A genetic match was confirmed between the HRM curves obtained from the mycelia (evidence) and biological tissue extracted from the fruiting bodies. Mycelia recovered from the evidence and fruiting bodies (control) were genetically indistinguishable.

Whole genome sequence of Auricularia heimuer (Basidiomycota, Fungi), the third most important cultivated mushroom worldwide.

Heimuer, Auricularia heimuer, is one of the most famous traditional Chinese foods and medicines, and it is the third most important cultivated mushroom worldwide. The aim of this study is to develop genomic resources for A. heimuer to furnish tools that can be used to study its secondary metabolite production capability, wood degradation ability and biosynthesis of polysaccharides. The genome was obtained from single spore mycelia of the strain Dai 13782 by using combined high-throughput Illumina HiSeq 4000 system with the PacBio RSII long-read sequencing platform. Functional annotation was accomplished by blasting protein sequences with different public available databases to obtain their corresponding annotations. It is 49.76Mb in size with a N50 scaffold size of 1,350,668bp and encodes 16,244 putative predicted genes. This is the first genome-scale assembly and annotation for A. heimuer, which is the third sequenced species in Auricularia.

New host records of three Kudoa spp. (K. yasunagai, K. thalassomi, and K. igami) with notable variation in the number of shell valves and polar capsules in spores.

To date, 26 Kudoa spp. (Myxozoa: Myxosporea: Multivalvulida) have been recorded in edible marine fishes in Japan. In the future, it is likely that even more marine fish multivalvulid myxosporeans will be characterized morphologically and genetically, which will aid the precise understanding of their biodiversity and biology. We examined 60 individuals of six fish species collected from the Philippine Sea off Kochi or from the border between the Philippine Sea and East China Sea around Miyako Island, Okinawa, i.e., the southern part of Japan. Newly collected parasite species included Kudoa yasunagai from the brain of Japanese meagre (Argyrosomus japonicus) and Japanese parrotfish (Calotomus japonicus), Kudoa miyakoensis n. sp. and Kudoa thalassomi from the brain and trunk muscle, respectively, of bluespine unicornfish (Naso unicornis), and Kudoa igami from the trunk muscle of Carolines parrotfish (Calotomus carolinus), African coris (Coris gaimard), and Pastel ringwrasse (Hologymnosus doliatus). With the exception of Japanese parrotfish for K. yasunagai, all these fish are new host records for each kudoid species. Notable variation in the number of shell valves (SV) and polar capsules (PC) was observed for all four kudoid species. In particular, spores with seven or eight SV/PC were prominent in K. igami isolates, despite the original Japanese parrotfish-derived description characterizing it as having spores with six, or less commonly five, SV/PC. However, molecular genetic characterization based on the ribosomal RNA gene (rDNA) and mitochondrial DNA (cytochrome c oxidase subunit 1 and ribosomal RNA small and large subunits) found no significant differences in the nucleotide sequences of isolates with different phenotypical features as far as examined in the present study. A newly erected species, K. miyakoensis n. sp., was determined to be phylogenetically closest to brain-parasitizing species, such as K. chaetodoni, K. lemniscati, and K. yasunagai based on rDNA nucleotide sequences, but differed from them morphologically.

Proline synthesis in developing microspores is required for pollen development and fertility.

BACKGROUND: In many plants, the amino acid proline is strongly accumulated in pollen and disruption of proline synthesis caused abortion of microspore development in Arabidopsis. So far, it was unclear whether local biosynthesis or transport of proline determines the success of fertile pollen development. RESULTS: We analyzed the expression pattern of the proline biosynthetic genes PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 & 2 (P5CS1 & 2) in Arabidopsis anthers and both isoforms were strongly expressed in developing microspores and pollen grains but only inconsistently in surrounding sporophytic tissues. We introduced in a p5cs1/p5cs1 p5cs2/P5CS2 mutant background an additional copy of P5CS2 under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, the tapetum-specific LIPID TRANSFER PROTEIN 12 (Ltp12) promoter or the pollen-specific At5g17340 promoter to determine in which site proline biosynthesis can restore the fertility of proline-deficient microspores. The specificity of these promoters was confirmed by ß-glucuronidase (GUS) analysis, and by direct proline measurement in pollen grains and stage-9/10 anthers. Expression of P5CS2 under control of the At5g17340 promoter fully rescued proline content and normal morphology and fertility of mutant pollen. In contrast, expression of P5CS2 driven by either the Ltp12 or CaMV35S promoter caused only partial restoration of pollen development with little effect on pollen fertility. CONCLUSIONS: Overall, our results indicate that proline transport is not able to fulfill the demand of the cells of the male germ line. Pollen development and fertility depend on local proline biosynthesis during late stages of microspore development and in mature pollen grains.

Morphology and 18S rDNA sequencing of Henneguya peruviensis n. sp. (Cnidaria: Myxosporea), a parasite of the Amazonian ornamental fish Hyphessobrycon loretoensis from Peru: A myxosporean dispersal approach.

Myxosporean are endoparasitic cnidarians of wide distribution and responsible for important economic losses in fisheries and aquaculture. A new myxosporean species, Henneguya peruviensis n. sp., is herein described as obtained from the gill filaments of Hyphessobrycon loretoensis caught in the Nanay River, Department of Loreto, Peru. The parasite was found in 37 of 45 (82.2%) examined H. loretoensis. The new species was characterized based on morphological features and 18S rDNA gene sequence data. The sequencing of the 18S rDNA gene from the spores of H. peruviensis n. sp. resulted in 1632 nucleotides and this sequence did not match any of the myxozoan available in the GenBank. Phylogenetic analysis showed that H. peruviensis n. sp. closed together with H. leporinicola. Nonetheless, the 18S rDNA sequences of H. peruviensis n. sp. and H. leporinicola have only 82% similarity. This is the first description and molecular study of a Myxozoa parasitizing fish of the genus Hyphessobrycon in the Amazon basin. Given the importance of the ornamental fish industry in translocation of aquatic organisms worldwide, the international movement of myxosporeans in infected fish is discussed in terms of disease outbreaks and the need for preventative action.

Myxobolus infection in the cornea of the roach (Rutilus rutilus) in Lake Balaton.

Infection of the cornea in fishes by Myxobolus plasmodia is a common but still little known site preference of myxosporeans. A sporadic but striking infection in the cornea of the roach (Rutilus rutilus) was observed in Lake Balaton, Hungary. Relatively small, round plasmodia 250 to 500 µm in diameter developed in the dense connective tissue of the cornea. Morphological and molecular biological examination of spores collected from cysts in the cornea demonstrated that this infection is caused by Myxobolus fundamentalis, a species hitherto reported only from the cartilaginous gill arch of the roach. The 18S rDNA sequences of spores from the cornea showed 99.9% identity to the sequences of spores from the gill arch, and they also shared 99.9% identity with the sequences of triactinomyxon actinospores obtained from the oligochaete Isochaetides michaelseni.

Quorum sensing in rhizobia isolated from the spores of the mycorrhizal symbiont Rhizophagus intraradices.

Most beneficial services provided by arbuscular mycorrhizal fungi (AMF), encompassing improved crop performance and soil resource availability, are mediated by AMF-associated bacteria, showing key-plant growth-promoting (PGP) traits, i.e., the production of indole acetic acid, siderophores and antibiotics, and activities increasing the availability of plant nutrients by nitrogen fixation and phosphate mobilization. Such functions may be affected by the ability of AMF-associated bacteria to communicate through the production and secretion of extracellular small diffusible chemical signals, N-acyl homoserine lactone signal molecules (AHLs), that regulate bacterial behavior at the community level (quorum sensing, QS). This work investigated the occurrence and extent of QS among rhizobia isolated from AMF spores, using two different QS reporter strains, Agrobacterium tumefaciens NTL4 pZRL4 and Chromobacterium violaceum CV026. We also assessed the quorum quenching (QQ) activity among Bacillus isolated from the same AMF spores. Most rhizobia were found to be quorum-signaling positive, including six isolates producing very high levels of AHLs. The results were confirmed by microtiter plate assay, which detected 65% of the tested bacteria as medium/high AHL producers. A 16S rDNA sequence analysis grouped the rhizobia into two clusters, consistent with the QS phenotype. None of the tested bacteria showed QQ activity able to disrupt the QS signaling, suggesting the absence of antagonism among bacteria living in AMF sporosphere. Our results provide the first evidence of the ability of AMF-associated rhizobia to communicate through QS, suggesting further studies on the potential importance of such a behavior in association with key-plant growth-promoting functions.