Nutritional symbionts of a putative vector, <Emphasis Type="Italic">Xyleborus bispinatus</Emphasis>, of the laurel wilt pathogen of avocado, <Emphasis Type="Italic">Raffaelea lauricola</Emphasis>

Ambrosia beetles subsist on fungal symbionts that they carry to, and cultivate in, their natal galleries. These symbionts are usually saprobes, but some are phytopathogens. Very few ambrosial symbioses have been studied closely, and little is known about roles that phytopathogenic symbionts play in the life cycles of these beetles. One of the latter symbionts, Raffaelea lauricola, causes laurel wilt of avocado, Persea americana, but its original ambrosia beetle partner, Xyleborus glabratus, plays an uncertain role in this pathosystem. We examined the response of a putative, alternative vector of R. lauricola, Xyleborus bispinatus, to artificial diets of R. lauricola and other ambrosia fungi. Newly eclosed, unfertilized females of X. bispinatus were reared in no-choice assays on one of five different symbionts or no symbiont. Xyleborus bispinatus developed successfully on R. lauricola, R. arxii, R. subalba and R. subfusca, all of which had been previously recovered from field-collected females of X. bispinatus. However, no development was observed in the absence of a symbiont or on another symbiont, Ambrosiella roeperi, recovered from another ambrosia beetle, Xylosandrus crassiusculus. In the no-choice assays, mycangia of foundress females of X. bispinatus harbored significant colony-forming units of, and natal galleries that they produced were colonized with, the respective Raffaelea symbionts; with each of these fungi, reproduction, fecundity and survival of the beetle were positively impacted. However, no fungus was recovered from, and reproduction did not occur on, the A. roeperi and no symbiont diets. These results highlight the flexible nature of the ambrosial symbiosis, which for X. bispinatus includes a fungus with which it has no evolutionary history. Although the “primary” symbiont of the neotropical X. bispinatus is unclear, it is not the Asian R. lauricola.     

Genetic diversity of free-living <Emphasis Type="Italic">Symbiodinium</Emphasis> in the Caribbean: the importance of habitats and seasons

Although reef corals are dependent of the dinoflagellate Symbiodinium, the large majority of corals spawn gametes that do not contain their vital symbiont. This suggests the existence of a pool of Symbiodinium in the environment, of which surprisingly little is known. Reefs around Curaçao (Caribbean) were sampled for free-living Symbiodinium at three time periods (summer 2009, summer 2010, and winter 2010) to characterize different habitats (water column, coral rubble, sediment, the macroalgae Halimeda spp., Dictyota spp., and Lobophora variegata, and the seagrass Thalassia testudinum) that could serve as environmental sources of symbionts for corals. We detected the common clades of Symbiodinium that engage in symbiosis with Caribbean coral hosts A, B, and C using Symbiodinium-specific primers of the hypervariable region of the chloroplast 23S ribosomal DNA gene. We also discovered clade G and, for the first time in the Caribbean, the presence of free-living Symbiodinium clades F and H. Additionally, this study expands the habitat range of free-living Symbiodinium as environmental Symbiodinium was detected in T. testudinum seagrass beds. The patterns of association between free-living Symbiodinium types and habitats were shown to be complex. An interesting, strong association was seen between some clade A sequence types and sediment, suggesting that sediment could be a niche where clade A radiated from a free-living ancestor. Other interesting relationships were seen between sequence types of Symbiodinium clade C with Halimeda spp. and clades B and F with T. testudinium. These relationships highlight the importance of some macroalgae and seagrasses in hosting free-living Symbiodinium. Finally, studies spanning beyond a 1-yr cycle are needed to further expand on our results in order to better understand the variation of Symbiodinium in the environment through time. All together, results presented here showed that the great diversity of free-living Symbiodinium has a dynamic distribution across habitats and time.     

Molecular evidence of symbiotic activity between <Emphasis Type="Italic">Symbiodinium</Emphasis> and <Emphasis Type="Italic">Tridacna maxima</Emphasis> larvae

Dinoflagellates in the genus Symbiodinium (zooxanthellae) provide the photosynthesis that sustains the majority of primary production in coral reefs. They occur symbiotically with several phyla, including mollusks such as giant clams (Tridacna spp.). This mutualistic association is obligatory for the giant clams, but the exact point in which this symbiosis is established and the main translocated photosynthate are unknown. In this study, we tracked the expression of specific genes for symbiosis and glycerol synthesis during a time course experiment. Giant clam larvae were raised until 75 h post-fertilization and then infected with cultured isolates of Symbiodinium clade A3. Expression of symbiosis-specific and housekeeping genes was monitored at four time points. The expression of H⁺-ATPase, a symbiosis-specific gene in Symbiodinium, was observed at 24 h after symbiont acquisition by the clam larvae. The expression of an enzyme responsible for glycerol synthesis was also observed. Together, these results show that the symbiotic relationship was already in place 24 h after Symbiodinium acquisition, during veliger larval stage. This is the first report using a molecular symbiosis-specific marker that supports symbiotic activity between Symbiodinium and a metazoan larva of an organism that acquires symbionts horizontally. From the expression of the glycerol-synthesizing gene, it was qualitatively determined that Symbiodinium cells may produce glycerol regardless of whether they are free-living or in symbiosis.     

Trans-generational specificity within a cnidarian–algal symbiosis

Ocean warming and other anthropogenic stresses threaten the symbiosis between tropical reef cnidarians and their dinoflagellate endosymbionts (Symbiodinium). Offspring of many cnidarians acquire their algal symbionts from the environment, and such flexibility could allow corals to respond to environmental changes between generations. To investigate the effect of both habitat and host genotype on symbiont acquisition, we transplanted aposymbiotic offspring of the common Caribbean octocoral Briareum asbestinum to (1) an environmentally different habitat that lacked B. asbestinum and (2) an environmentally similar habitat where local adults harbored Symbiodinium phylotypes that differed from parental colonies. Symbiont acquisition and establishment of symbioses over time was followed using a within-clade DNA marker (23S chloroplast rDNA) and a within-phylotype marker (unique alleles at a single microsatellite locus). Early in the symbiosis, B. asbestinum juveniles harbored multiple symbiont phylotypes, regardless of source (parent or site). However, with time (~4 yr), offspring established symbioses with the symbiont phylotype dominant in the parental colonies, regardless of transplant location. Within-phylotype analyses of the symbionts revealed a similar pattern, with offspring acquiring the allelic variant common in symbionts in the parental population regardless of the environment in which the offspring was reared. These data suggest that in this host species, host–symbiont specificity is a genetically determined trait. If this level of specificity is widespread among other symbiotic cnidarians, many cnidarian–algal symbioses may not be able to respond to rapid, climate change-associated environmental changes by means of between-generation switching of symbionts.     

Arsenic methylation by a genetically engineered <Emphasis Type="Italic">Rhizobium</Emphasis>-legume symbiont

Background and aims

Arsenic (As) is one of the most widespread environmental contaminants. The aim of our study was to test a novel bioremediation system based on the symbiosis between leguminous plant and genetically engineered rhizobia.

Methods

The arsenite [As(III)] S-adenosylmethionine methyltransferase gene (CrarsM) from the alga Chlamydomonas reinhardtii was inserted into the chromosome of Rhizobium leguminosarum bv. trifolii strain R3. The As methylation ability of the recombinant Rhizobium was tested under free living conditions and in symbiosis with red clover plants. Arsenic speciation was determined using high-performance liquid chromatography-inductively coupled plasma mass spectrometry.

Results

Under free-living conditions, CrarsM-recombinant R. leguminosarum gained the ability to methylate As(III) to methylated arsenicals, including methylarsenate [MAs(V)], dimethylarsenate [DMAs(V)] and trimethylarsine oxide [TMAs(V)O]. Red clover plants were inoculated with either control (non-recombinant) or CrarsM-recombinant R. leguminosarum and exposed to 5 or 10 μM arsenite. No methylated As species were detected in red clover plants inoculated with control R. leguminosarum. In contrast, all three methylated species were detected in both the nodules and the shoots when the recombinant Rhizobium established symbiosis with red clover, accounting for 74.7–75.1% and 29.1–42.4% of the total As in the two plant tissues, respectively. The recombinant symbiont also volatilized small amounts of As.

Conclusions

The present study demonstrates that engineered rhizobia expressing an algal arsM gene can methylate and volatilize As, providing a proof of concept for potential future use of legume-rhizobia symbionts for As bioremediation.

     

Diversity of the Most Commonly Reported Facultative Symbionts in Two Closely-Related Aphids with Different Host Ranges

Richness and abundance of facultative symbionts vary strongly with aphid species and genotype, symbiont strain, host plant, biogeography, and a number of abiotic factors. Despite indications that aphids in the same ecological niche show similar levels of facultative symbiont richness, existing reports do not consider the potential role of host plants on aphid microbial community. Little is known about how oligophagy and polyphagy may be influenced by secondary symbiont distribution, mainly because studies on secondary symbiont diversity are biased towards polyphagous aphids from the Northern Hemisphere. Here, we demonstrate the richness and abundance of the most common aphid-associated facultative symbionts in two tropical aphid species, the oligophagous Aphis (Toxoptera) citricidus (Kirkaldy) (Hemiptera: Aphididae) and the polyphagous Aphis aurantii (Boyer de Fonscolombe) (Hemiptera: Aphididae). Aphis citricidus is restricted to Citrus sp. host plants and closely related genera, whereas A. aurantii successfully exploits a wide variety of host plants from different families. Both were collected in the same ecological niche and our data basically indicated the same richness of secondary symbionts, but the abundance at which secondary symbionts occurred was very distinct between the two species. Spiroplasma was the most abundant facultative symbiont associated with A. citricidus and A. aurantii in the ecological niche studied. Single and multiple secondary symbiont infections were observed, but diversity of multiple infections was particularly high in A. citricidus. We discuss our findings and suggest hypotheses to explain causes and consequences of the differences in secondary symbiont diversity observed between these two aphid species.     

Aseptic rearing procedure for the stinkbug <Emphasis Type="Italic">Plautia stali</Emphasis> (Hemiptera: Pentatomidae) by sterilizing food-derived bacterial contaminants

The stinkbug Plautia stali Scott is a notorious agricultural pest whose posterior midgut hosts specific bacteria essential for its growth and survival, highlighted as an experimental model for symbiosis studies. Some symbiotic bacteria of P. stali are cultivable, found free-living in and acquired from the environment, and, furthermore, some free-living environmental bacteria are potentially capable of establishing symbiotic association with P. stali. In this context, it is expected that such environmental bacteria may occasionally contaminate and infect the experimental insects maintained in the laboratory, which could potentially affect the functional analyses of the symbiosis. Here we report that such contamination events do occur under a laboratory rearing conditions for P. stali. When symbiont-deprived newborn nymphs from surface-sterilized eggs were reared in sterilized plastic containers with autoclaved water, most of them died as nymphs presumably as a result of aposymbiosis, but only a small fraction could attain adulthood and the adult insects were all infected with γ-proteobacteria allied to Pantoea and Enterobacter. A variety of bacteria, mainly Bacillus and also Pantoea and Enterobacter, were detected from peanuts and soybeans provided as food for P. stali. Autoclaving of peanuts and soybeans eradicated these bacteria but negatively affected the host survival, whereas ethanol sterilization of peanuts and soybeans removed Pantoea and Enterobacter, but not Bacillus, without negative effects on the host survival. On the basis of these results, we established a practical procedure for aseptic rearing of P. stali, which will enable reliable and strict analyses of host–symbiont interactions in the model symbiotic system.     

Interseasonal and interspecies diversities of <Emphasis Type="Italic">Symbiodinium</Emphasis> density and effective photochemical efficiency in five dominant reef coral species from Luhuitou fringing reef,northern South China Sea

Although it is well established that different coral species have different susceptibilities to thermal stress, the reasons behind this variation are still unclear. In this study, 384 samples across five dominant coral species were collected seasonally between September 2013 and August 2014 at Luhuitou fringing reef in Sanya, Hainan Island, northern South China Sea, and their algal symbiont density and effective photochemical efficiency (Φ _PSII) were measured. The results indicated that both the Symbiodinium density and Φ _PSII of corals were subject to significant interspecies and seasonal variations. Stress-tolerant coral species, including massive Porites lutea and plating Pavona decussata, had higher symbiont densities but lower Φ _PSII compared to the vulnerable branching species of Acropora over the course of all four seasons. Seasonally, coral symbiont densities were the lowest during winter, while during the same period, Φ _PSII of corals was at the highest point. Further analysis suggested that dissolved inorganic nutrients and upwelling in the reef area were probably responsible for the observed seasonal variations in symbiont density. The fact that Porites lutea has the lowest Φ _PSII during all four seasons is likely related to their symbionts’ lower capacity to provide required photosynthates for calcification. These results suggest that a coral’s thermal tolerance is primarily and positively dependent on its symbiont density and is less related to its effective photochemical efficiency.     

Diversity in symbiont consortia in the pea aphid complex is associated with large phenotypic variation in the insect host

Virtually all eukaryotes host microbial symbionts that influence their phenotype in many ways. In a host population, individuals may differ in their symbiotic complement in terms of symbiont species and strains. Hence, the combined expression of symbiont and host genotypes may generate a range of phenotypic diversity on which selection can operate and influence host population ecology and evolution. Here, we used the pea aphid to examine how the infection with various symbiotic complements contributes to phenotypic diversity of this insect species. The pea aphid hosts an obligate symbiont (Buchnera aphidicola) and several secondary symbionts among which is Hamiltonella defensa. This secondary symbiont confers a protection against parasitoids but can also reduce the host’s longevity and fecundity. These phenotypic effects of H. defensa infection have been described for a small fraction of the pea aphid complex which encompasses multiple plant-specialized biotypes. In this study, we examined phenotypic differences in four pea aphid biotypes where H. defensa occurs at high frequency and sometimes associated with other secondary symbionts. For each biotype, we measured the fecundity, lifespan and level of parasitoid protection in several aphid lineages differing in their symbiotic complement. Our results showed little variation in longevity and fecundity among lineages but strong differences in their protection level. These differences in protective levels largely resulted from the strain type of H. defensa and the symbiotic consortium in the host. This study highlights the important role of symbiotic complement in the emergence of phenotypic divergence among host populations of the same species.     

Various <Emphasis Type="Italic">Wolbachia</Emphasis> genotypes differently influence host <Emphasis Type="Italic">Drosophila</Emphasis> dopamine metabolism and survival under heat stress conditions

Background

One of the most widespread prokaryotic symbionts of invertebrates is the intracellular bacteria of Wolbachia genus which can be found in about 50% of insect species. Wolbachia causes both parasitic and mutualistic effects on its host that include manipulating the host reproductive systems in order to increase their transmission through the female germline, and increasing the host fitness. One of the mechanisms, promoting adaptation in biological organisms, is a non-specific neuroendocrine stress reaction. In insects, this reaction includes catecholamines, dopamine, serotonin and octopamine, which act as neurotransmitters, neuromodulators and neurohormones. The level of dopamine metabolism correlates with heat stress resistance in Drosophila adults.

Results

To examine Wolbachia effect on Drosophila survival under heat stress and dopamine metabolism we used five strains carrying the nuclear background of interbred Bi90 strain and cytoplasmic backgrounds with different genotype variants of Wolbachia (produced by 20 backcrosses of Bi90 males with appropriate source of Wolbachia). Non-infected Bi90 strain (treated with tetracycline for 3 generations) was used as a control group. We demonstrated that two of five investigated Wolbachia variants promote changes in Drosophila heat stress resistance and activity of enzymes that produce and degrade dopamine, alkaline phosphatase and dopamine-dependent arylalkylamine N-acetyltransferase. What is especially interesting, wMelCS genotype of Wolbachia increases stress resistance and the intensity of dopamine metabolism, whereas wMelPop strain decreases them. wMel, wMel2 and wMel4 genotypes of Wolbachia do not show any effect on the survival under heat stress or dopamine metabolism. L-DOPA treatment, known to increase the dopamine content in Drosophila, levels the difference in survival under heat stress between all studied groups.

Conclusions

The genotype of symbiont determines the effect that the symbiont has on the stress resistance of the host insect.

     

Obligate gut symbiotic association in the sloe bug <Emphasis Type="Italic">Dolycoris baccarum</Emphasis> (Hemiptera: Pentatomidae)

A number of phytophagous stinkbugs are associated with specific bacterial symbionts in their alimentary tracts. The sloe bug Dolycoris baccarum (Linnaeus), a notorious pest of diverse crops, possesses a number of sac-like tissues, called crypts, in a posterior section of the midgut, wherein a specific bacterial symbiont colonizes. Here we characterized the symbiotic bacterium of D. baccarum by histological analysis, molecular phylogeny, and diagnostic PCR with a specific primer set. The cloning and sequencing analyses of bacterial 16S rRNA genes and fluorescent in situ hybridization demonstrated that the sloe bug is associated with a single species of Gammaproteobacteria in the midgut crypts. Molecular phylogenetic analysis strongly suggested that the symbiont should be placed in the genus Pantoea of the Enterobacteriaceae. Diagnostic PCR and egg surface sterilization with formalin indicated the stinkbug vertically transmits the Pantoea symbiont via egg-smearing. The sterilization-produced aposymbiotic nymphs showed high mortality and no insects reached adulthood. In addition, the Pantoea symbiont was uncultivable outside the insect host, indicating an obligate and intimate host-symbiont association.     

<Emphasis Type="Italic">Entransia</Emphasis> and <Emphasis Type="Italic">Hormidiella</Emphasis>, sister lineages of <Emphasis Type="Italic">Klebsormidium</Emphasis> (Streptophyta), respond differently to light,temperature, and desiccation stress

The green-algal class Klebsormidiophyceae (Streptophyta), which occurs worldwide, comprises the genera Klebsormidium, Interfilum, Entransia, and Hormidiella. Ecophysiological research has so far focused on the first two genera because they are abundant in biological soil crust communities. The present study investigated the photosynthetic performances of Hormidiella attenuata and two strains of Entransia fimbriata under light, temperature, and desiccation stress. Their ultrastructure was compared using transmission electron microscopy. The two Entransia strains showed similar physiological responses. They used light more efficiently than Hormidiella, as indicated by higher oxygen production and relative electron transport rate under low light conditions, lower light saturation and compensation points, and higher maximum oxygen production during light saturation. Their requirement for low light levels explains the restriction of Entransia to dim limnetic habitats. In contrast, Hormidiella, which prefers drier soil habitats, responded to light gradients similarly to other aero-terrestrial green algae. Compared to Entransia, Hormidiella was less affected by short-term desiccation, and rehydration allowed full recovery of the photosynthetic performance. Nevertheless, both strains of Entransia coped with low water availability better than other freshwater algae. Photosynthetic oxygen production in relation to respiratory consumption was higher in low temperatures (Entransia: 5 °C, Hormidiella: 10 °C) and the ratio decreased with increasing temperatures. Hormidiella exhibited conspicuous triangular spaces in the cell wall corners, which were filled either with undulating cell wall material or with various inclusions. These structures are commonly seen in various members of Klebsormidiophyceae. The data revealed significant differences between Hormidiella and Entransia, but appropriate adaptations to their respective habitats.     

Quantitative criteria for estimating the effectiveness of bioluminescence expression in natural and transgenic luminescent bacteria

Computational coefficients for estimating the effectiveness of bioluminescence expression in natural luminescent bacteria Photobacterium leiognathi 54 and transgenic strain E. coli Z905/pPHL7 bearing lux-operon in a multicopy plasmid are suggested and their use at molecular, cell, and population levels was considered. It was shown that at the population level, all transgenic variants have an advantage over natural variants of P. leiognathi 54 irrespective of the type of lux-operon regulation. At the cell level, the effectiveness of bioluminescence expression in the bright and dim variants of the transgenic strain increased by several orders. At the level of one lux-operon, the effectiveness of expression in the bright variant of the transgenic strain is substantially higher than in the natural bright variant; in dim variants, the efficiency values are similar; the effectiveness of bioluminescence expression in the dark variant of E. coli Z905-2/pPHL7 is by two orders of magnitude lower than in the dark variant of P. leiognathi 54.     

Detection of symbionts and virus in the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis> (Hemiptera: Aleyrodidae), vector of the <Emphasis Type="Italic">Mungbean yellow mosaic India virus</Emphasis> in Central India

Legume crops in Central India, the main soybean production area of the country, may suffer from yellow mosaic disease caused by the Mungbean yellow mosaic India virus (MYMIV). MYMIV is transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), which is a species complex composed of various genetic groups. This vector species harbors different endosymbionts among regional strains and among individuals. To elucidate fundamental aspects of this virus vector in the state of Madhya Pradesh, the infection status of the symbionts and the virus in whiteflies was studied. A polymerase chain reaction (PCR) survey of the whiteflies collected in Madhya Pradesh found four secondary endosymbionts, Arsenophonus, Hemipteriphilus, Wolbachia, and Cardinium, in addition to the primary endosymbiont Portiera. Arsenophonus and Hemipteriphilus were highly infected but the infection rates of Wolbachia and Cardinium were low. MYMIV was detected in whitefly populations collected from various host plants in Madhya Pradesh. The whitefly populations belonged to the Asia I and II genetic groups; several different Asia II populations were also distributed. Specific relations were not observed among symbiont infection status, virus infection, and the whitefly genetic groups in the populations of Madhya Pradesh, though Cardinium was highly detected in the Asia II-1 group. New primers, which can be used for PCR template validation and for discriminating two phylogenetically close endosymbionts, were designed.     

The potential of azooxanthellate poriferan hosts to assess the fundamental and realized <Emphasis Type="Italic">Symbiodinium</Emphasis> niche: evaluating a novel method to initiate <Emphasis Type="Italic">Symbiodinium</Emphasis> associations

On coral reefs, Symbiodinium spp. are found in most cnidarian species, but reside in only a small number of sponge species. Of the sponges that do harbor Symbiodinium, most are found in the family Clionaidae, which represents a minor fraction of the poriferan diversity on a reef. Our goal was to determine whether Symbiodinium can be taken up by sponge hosts that do not typically harbor these algal symbionts, and then to follow the fate of any Symbiodinium that enter the intracellular space. We used the filter-feeding capacity of sponges to initiate intracellular interactions between sponge-specialist clade G Symbiodinium and six sponge species that do not associate with Symbiodinium. Using a pulse-chase experimental design, we determined that all of the species we examined captured Symbiodinium, and undamaged intracellular algae were found up to 1 h after inoculation. In a longer-term experiment, Symbiodinium populations in Amphimedon erina persisted in sponge cells for at least 5 d post-inoculation. While no evidence of digestion was detected, the population decreased exponentially after inoculation. We contrast these data with the characteristics of symbiont acquisition and establishment in Cliona varians, which normally harbors Symbiodinium. Explants from experimentally derived aposymbiotic sponges were placed in the field where they acquired Symbiodinium from ambient sources (i.e., we did not inoculate them as in the pulse-chase experiments). We began to detect Symbiodinium cells in C. varians after 12 d, and the algal population increased exponentially until densities approached those typically found in this host (after ~128 d). We discuss the implications of this work in light of growing interest in the evolution of specificity between hosts and symbionts, and the fundamental and realized niche of Symbiodinium.     

Molecular authentication of two medicinal plants <Emphasis Type="Italic">Ligularia fischeri</Emphasis> and <Emphasis Type="Italic">Ligularia stenocephala</Emphasis> using allele-specific PCR (AS-PCR) strategy

Ligularia fischeri (Gom-chi) and Ligularia stenocephala (Gon-dal-bi) are popular edible herbs in Korea. L. fischeri is used to treat jaundice, hepatitis, rheumatoid arthritis, and scarlet fever, while L. stenocephala is used to treat anxiety, weakness, and menstrual disorders. The herbal medicinal activities of these two herbs differ, but they are very difficult to distinguish based on their morphologies, especially in their dried forms. In an effort to distinguish these two plant species, we sequenced three barcoding genes in plastids, matK, rbcL, and trnH-psbA. From the analysis of sequence variations, we detected five single nucleotide polymorphisms (SNPs) between two the species. Allele specific (AS)-primers in the SNPs were employed in discrimination of the two species. Of the five AS-primer sets, one primer pair in matK gene showed reproducibly distinguishable PCR amplification between plants of L. fischeri and L. stenocephala. The method is reproducible and efficient, and is the first reported molecular method to discriminate between L. fischeri and L. stenocephala.     

The Melanin Biosynthesis Gene from the CA15-1 Strain of Alfalfa Nodule Bacteria: Molecular Analysis and Phylogeny

Identification and analysis of the melanin biosynthesis gene in the Sinorhizobium meliloti CA15-1 strain were carried out. Tn5 mutants, which lost the ability to synthesize melanin, were obtained. Molecular biological analysis of the mepA gene encoding tyrosinase was performed. In contrast to other members of the Rhizobiacea group, the identical structure of the mepA locus was revealed for all strains of nodule bacteria of the Sinorhizobium genus. Phylogenetic analysis indicated that horizontal transfer of the mepA gene occurred in the course of evolution of bacterial tyrosinases. At the same time, the closely related members of nodule bacteria “acquired” this gene from different sources. Analysis of symbiotic properties of the Mep^– mutants of the CA15-1 strain showed that melanin did not affect the ability to go into an efficient symbiosis with alfalfa. Most probably, it is important only at the stages of adaptation of the free-living cells in the environment.