Increased hyphal branching and growth of ectomycorrhizal fungus Lactarius rufus by the helper bacterium Paenibacillus sp

Paenibacillus sp. EJP73 has been previously demonstrated as a mycorrhization helper bacterium (MHB) for the Lactarius rufus–Pinus sylvestris symbiosis in both laboratory and glasshouse experiments. In the present study, the effect of Paenibacillus sp. EJP73 metabolites on L. rufus EO3 pre-symbiotic growth was tested in two agar plate-based systems. Specifically, volatile metabolites were investigated using a dual plate system, in which the presence of strain EJP73 resulted in a significant negative effect on L. rufus EO3 hyphal radial growth but enhanced hyphal branching and reduced internode distance. Soluble metabolites produced by strain EJP73 were tested on L. rufus EO3 growth in single-agar plate assays by incorporating bacterial cell-free whole or molecular weight fraction spent broth into the agar. Whole spent broth had a negative effect on hyphal growth, whereas a low molecular weight fraction (100–1,000) promoted colony radial growth. Headspace and spent broth analysis of strain EJP73 cultures revealed 2,5-diisopropylpyrazine to be the most significant component. Synthesised 2,5-diisopropylpyrazine and elevated CO₂ (2,000 ppm) were tested as specific volatile metabolites in the dual plate system, but neither produced the response shown when strain EJP73 was present. Increased pre-symbiotic hyphal branching leading to increased likelihood of plant infection may be an important MHB mechanism for strain EJP73. Although the precise signal molecules could not be identified, the work suggests a number of metabolites may work synergistically to increase L. rufus root colonisation.     

Location and Survival of Mycorrhiza Helper Pseudomonas fluorescens during Establishment of Ectomycorrhizal Symbiosis between Laccaria bicolor and Douglas Fir

The mycorrhiza helper bacterium Pseudomonas fluorescens BBc6, isolated from a Laccaria bicolor sporocarp, consistently promotes L. bicolor-Douglas fir (Pseudotsuga menziesii) ectomycorrhizal formation, even with low doses of bacterial inoculum. In order to describe this phenomenon more accurately, we have looked at the location and survival of the introduced bacterial strain in the soil and in the rhizosphere during the establishment of mycorrhizal symbiosis in glasshouse and nursery experiments. Bacterial populations were quantified with a spontaneous, stable, rifampin-resistant mutant, BBc6R8, which phenotypically conformed to the parental strain. BBc6R8 populations declined rapidly, reaching the detection limit after 19 weeks, and did not increase either when L. bicolor sporocarps were forming in autumn or when Douglas fir roots resumed growing in spring. BBc6R8 was neither an endophyte nor a rhizobacterium. Furthermore, it was not particularly associated with either mycorrhizas of Douglas fir-L. bicolor or L. bicolor sporocarps. Surprisingly, a significant mycorrhiza helper effect was observed when the inoculated BBc6R8 population had dropped as low as 30 CFU g of dry matter(sup-1) in the soil. This study raises questions concerning the bacterial concentration in the soil which is effective for promotion of mycorrhizal establishment and the timing of the bacterial effect. It allows us to develop working hypotheses, which can be tested experimentally, to identify the mechanisms of the mycorrhiza helper effect.     

Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation

Mycorrhization helper bacteria (MHB), isolated from phylogenetically distinct ectomycorrhizal symbioses involving Lactarius rufus, Laccaria bicolor or Suillus luteus, were tested for fungus specificity to enhance L. rufus–Pinus sylvestris or L. bicolor–P. sylvestris mycorrhiza formation. As MHB isolated from the L. rufus and S. luteus mycorrhiza were originally characterised using a microcosm system, we assessed their ability to enhance mycorrhiza formation in a glasshouse system in order to determine the extent to which MHB are system-specific. Paenibacillus sp. EJP73, an MHB for L. rufus in the microcosm, significantly enhanced L. bicolor mycorrhiza formation in the glasshouse, demonstrating that the MHB effect of this bacterium is neither fungus-specific nor limited to the original experimental system. Although the five MHB strains studied were unable to significantly enhance L. rufus mycorrhiza formation, two of them did have a significant effect on dichotomous short root branching by L. rufus. The effect was specific to Paenibacillus sp. EJP73 and Burkholderia sp. EJP67, the two strains isolated from L. rufus mycorrhiza, and was not associated with auxin production. Altered mycorrhiza architecture rather than absolute number of mycorrhizal roots may be an important previously overlooked parameter for defining MHB effects.     

The mycorrhiza helper bacteria revisited

In natural conditions, mycorrhizal fungi are surrounded by complex microbial communities, which modulate the mycorrhizal symbiosis. Here, the focus is on the so-called mycorrhiza helper bacteria (MHB). This concept is revisited, and the distinction is made between the helper bacteria, which assist mycorrhiza formation, and those that interact positively with the functioning of the symbiosis. After considering some examples of MHB from the literature, the ecological and evolutionary implications of the relationships of MHB with mycorrhizal fungi are discussed. The question of the specificity of the MHB effect is addressed, and an assessment is made of progress in understanding the mechanisms of the MHB effect, which has been made possible through the development of genomics. Finally, clear evidence is presented suggesting that some MHB promote the functioning of the mycorrhizal symbiosis. This is illustrated for three critical functions of practical significance: nutrient mobilization from soil minerals, fixation of atmospheric nitrogen, and protection of plants against root pathogens. The review concludes with discussion of future research priorities regarding the potentially very fruitful concept of MHB.     

Stimulation of vesicular-arbuscular mycorrhiza by fungicides or rhizosphere bacteria

Stimulation of vesicular-arbuscular (VA) mycorrhizal fungi may secure the early establishment of symbiosis and benefit the host plant at an earlier stage of development. The application of Bacillus mycoides resulted in particular in the acceleration of early VA mycorrhiza formation. An increase in vigour of the symbiosis could be measured later in terms of increased sporulation of the mycorrhizal fungi after shoot removal. Natural sporulation during later mycorrhizal development was affected by combination of bacteria and just one mycorrhizal isolate. The stimulation of mycorrhizal development was shown to be non-specific with regard to host plant and the isolate of the VAM fungus. However, the effect could not be achieved in all combinations of soil types and host plants. Application of the systemic fungicides triadimefon and pyrazophos promoted VAM formation. Combinations of fungicide and bacterial treatments were not synergistic.     

Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans

Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi. Co-inoculation of soybean (Glycine max [L.] Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae. A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation. Inoculation with the mutant Rhizobium sp. NGR[delta]nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza. Highly purified Nod factors also increased the degree of mycorrhizal colonization. Nod factors from Rhizobium sp. NGR234 differed in their potential to promote fungal colonization. The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10-9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive. Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza. These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots.     

Production of staphylococcal enterotoxin in mixed cultures

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Production of staphylococcal enterotoxin in mixed cultures.

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Induction of glycyrrhizin and total phenolic compound production in licorice by using arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi have mutualistic symbiosis with higher plants, increasing plant resistance to environmental stresses and nutrient uptake and improving soil. During arbuscular mycorrhizal symbiosis, a range of chemical and biological factors are affected. In this study, two species of arbuscular mycorrhiza (Glomus mosseae and G. intraradices) were used to assess the effects of inoculation on licorice growth and secondary metabolite production. After successful inoculation, the increase in the growth rate, P and Zn uptake, and the accumulation of secondary metabolites in licorice (Glycyrrhiza glabra L.) roots were observed in two periods of 3 and 6 months compared to control. After 6 months, more increments in growth, secondary metabolites, and P and Zn uptake were observed compared with the first 3-months period. Two groups of secondary metabolites arising from phenolic and terpenoid metabolism obviously responded to mycorrhizal fungi colonization in licorice roots.     

Mycorrhization between <Emphasis Type="Italic">Cistus ladanifer</Emphasis> L. and <Emphasis Type="Italic">Boletus edulis</Emphasis> Bull is enhanced by the mycorrhiza helper bacteria <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Migula

Boletus edulis Bull. is one of the most economically and gastronomically valuable fungi worldwide. Sporocarp production normally occurs when symbiotically associated with a number of tree species in stands over 40 years old, but it has also been reported in 3-year-old Cistus ladanifer L. shrubs. Efforts toward the domestication of B. edulis have thus focused on successfully generating C. ladanifer seedlings associated with B. edulis under controlled conditions. Microorganisms have an important role mediating mycorrhizal symbiosis, such as some bacteria species which enhance mycorrhiza formation (mycorrhiza helper bacteria). Thus, in this study, we explored the effect that mycorrhiza helper bacteria have on the efficiency and intensity of the ectomycorrhizal symbiosis between C. ladanifer and B. edulis. The aim of this work was to optimize an in vitro protocol for the mycorrhizal synthesis of B. edulis with C. ladanifer by testing the effects of fungal culture time and coinoculation with the helper bacteria Pseudomonas fluorescens Migula. The results confirmed successful mycorrhizal synthesis between C. ladanifer and B. edulis. Coinoculation of B. edulis with P. fluorescens doubled within-plant mycorrhization levels although it did not result in an increased number of seedlings colonized with B. edulis mycorrhizae. B. edulis mycelium culture time also increased mycorrhization levels but not the presence of mycorrhizae. These findings bring us closer to controlled B. edulis sporocarp production in plantations.     

印度块菌(Tuber indicum)菌根促生细菌的研究

【目的】筛选对印度块菌菌根量和菌根苗长势有促进作用的菌根促生细菌(Mycorrhization helper bacteria,MHB)。【方法】选择华山松为宿主植物,自块菌菌根根际土壤中分离得到的11种细菌为供试菌株,将印度块菌菌剂与不同浓度的细菌混合于特定基质中后接种于华山松上,并通过对印度块菌与华山松形成的菌根数、华山松的株高和地径三方面的统计与分析,确认MHB。【结果】Pseudomonas sp. JCM 5481 (P143)、Streptomyces sp. EN31 (S191)、Variovorax paradoxus (V633)在浓度为2.4×109 CFU/mL时对印度块菌菌根数、株高和地径均有极显著促进作用(P<0.01);Pseudomonas chlororaphis (P11)、Pseudomonas corrugate (P127)在浓度为0.8×109 CFU/mL时对印度块菌菌根数、株高和地径均有显著促进作用(P<0.05)。4种假单胞菌浓度梯度的设置显示了不同菌株适宜的浓度不同。【结论】实验获得5种MHB,并表明细菌浓度是获得MHB的一个重要影响因素。     

THE ROLE OF ENDOMYCORRHIZAE IN REVEGETATION PRACTICES IN THE SEMI-ARID WEST. III. VERTICAL DISTRIBUTION OF VESICULAR-ARBUSCULAR (VA) MYCORRHIZA INOCULUM POTENTIAL

The changes in vesicular-arbuscular (VA) mycorrhiza inoculum potential (MIP) in soil profiles from a mid-elevation sage community were measured using a corn bioassay. The MIP was significantly reduced below 30 cm depth and approached zero at less than 1 m depth. The decrease in inoculum potential with depth in diluted soils did not always parallel changes in the nondiluted soil, indicating factors other than numbers of inoculum units also may be important in determining the extent of mycorrhiza formation. The relationship of these results to land disturbance and associated dilution of populations of VA mycorrhizal fungi and to defining topsoil is discussed.     

The effect of broth media on pneumococcal growth and the latex serotyping result

The aim was to test Todd-Hewitt broths (TH-broths) for the ability to propagate pneumococci and thereafter to evaluate the serotyping result obtained by the Pneumotest-Latex kit (SSI). TH-broths from four different producers (Oxoid, Sigma, Difco, and SSI) were tested and compared separately and with Serum broth (SSI). Twenty-three pneumococcal strains (different serotypes) were inoculated into the broths with start inoculums of 10(1), 10(3), and 10(6) CFU/ml. After incubation, overnight viable bacterial counts and visible growth were recorded. All pneumococci were serotyped with the Pneumotest-Latex kit. After incubation, the bacterial counts in all TH-broths were within the range of log 4.65-log 7.76 CFU/ml, while Serum broth showed an average growth ranging from log 8.05-log 8.90 CFU/ml. Comparing the growth of the four TH-broths showed no significant differences. In general, Serum broth had a more pronounced visual growth than each of the four TH-broths. Serotyping with Serum broth showed in general positive and correct latex typing results for all serotypes and initial inoculum, while the outcome of the TH-broths showed some false negative results depending on inoculum and serotype. Overall the Serum broth was found to be superior to the four TH-broths tested both with regard to CFU/ml and when used with the Pneumotest-Latex kit. However, if the Pneumotest-latex kit is only used on broths with visible growth as stated in the instruction manual, then the differences between the performances of the broths from the different producers was not significant and all broths could be used for Pneumotest-Latex typing.     

Reorganization of tobacco root plastids during arbuscule development

In the present paper we analyzed plastid populations labeled by the green fluorescent protein in non-mycorrhizal and mycorrhizal roots of tobacco (Nicotiana tahacum L.). We show by confocal laser scanning microscopy (i) a dramatic increase in these plastids in mycorrhizal roots and (ii) the formation of dense plastid networks covering the symbiotic interface of the arbuscular mycorrhiza, the arbuscule. These cytological observations point to an important role of root cortical cell plastids in the functioning of arbuscular mycorrhizal symbiosis.     

Differential effects of <Emphasis Type="Italic">Paenibacillus</Emphasis> spp. on cucumber mycorrhizas

The effects of 17 Paenibacillus strains on root colonization by Glomus intraradices or Glomus mosseae and plant growth parameters (shoot and root weight) of mycorrhizal cucumber plants were examined. The Paenibacillus strains were originally isolated from mycorrhizal (G. intraradices) and non-mycorrhizal cucumber rhizosphere and/or hyphosphere, except for strain EJP73, which originated from a Pinus sylvestris-Lactarius rufus ectomycorrhiza. Root colonization of cucumber plants by G. intraradices or G. mosseae was unaffected by all seven strains of Paenibacillus polymyxa, but was decreased or increased by four strains of Paenibacillus macerans and strain EJP73 of Paenibacillus sp. Overall, shoot dry weight of cucumber grown in symbioses with either G intraradices or G. mosseae was unaffected by inoculation with all of the Paenibacillus strains, except for strain MB02-429 of P. macerans, which increased the shoot dry weight in the cucumber-G. mosseae symbiosis. On the other hand, several Paenibacillus strains caused altered root growth. Three strains of P. polymyxa and four strains of P. macerans increased the root fresh weight of the cucumber–G. intraradices symbiosis, whereas three strains of P. polymyxa and one strain of P. macerans as well as Paenibacillus sp. EJP73, decreased the root fresh weight of the cucumber–G. mosseae symbiosis. In conclusion, our results show that bacteria from several species of Paenibacillus differentially affect cucumber mycorrhizas.     

Fungal selectivity of two mycorrhiza helper bacteria on five mycorrhizal fungi associated with <Emphasis Type="Italic">Pinus thunbergii</Emphasis>

The fungal selectivity of helper effect was revealed using four ectomycorrhizal fungi (Rhizopogon sp., Pisolithus tinctorius, Cenococcum geophilum and Suillus granulatus), and one ectoendomycorrhizal fungus (Wilcoxina mikolae). Previously, we isolated a rhizobacteria, Ralstonia basilensis and Bacillus subtilis, which have the ability to enhance the mycorrhizal symbiosis between S. granulatus and Pinus thunbergii. However, the characteristics of each bacterium on mycorrhizal fungi are still unclear. Therefore, we tried to examine the fungal selectivity of helper effect. A confrontation assay revealed that R. basilensis significantly promoted the in vitro hyphal growth of W. mikolae and S. granulatus, but it had no effect on Rhizopogon sp., P. tinctorius and C. geophilum. These results were consistent with the effects shown by R. basilensis on the mycorrhizal formation of these fungi. On the other hand, B. subtilis promoted the hyphal growth of W. mikolae and C. geophilum but suppressed growth of Rhizopogon sp. B. subtilis significantly stimulated the mycorrhizal formation of S. granulatus. Thus the effects of B. subtilis on hyphal growth and on mycorrhizal formation were inconsistent. These results suggest that R. basilensis and B. subtilis have fungal selective and different mechanisms in their roles as mycorrhiza helper bacteria.     

Mycorrhizal benefit in two low arctic herbs increases with increasing temperature

Climate change may influence the relationship between arctic plants and their symbiotic mycorrhizal fungi. The benefit of the symbiosis for the host plant affects vegetation succession and may be a key parameter in predicting vegetation responses to warming. We investigated the mycorrhizal benefit in the low arctic perennial herbs Potentilla crantzii and Ranunculus acris in symbiosis with the arbuscular mycorrhizal fungus Glomus claroideum. Temperature response in the mycorrhiza-mediated acquisition of nitrogen (N) and phosphorus (P), growth, and photosynthetic nutrient-use efficiency were determined. Near the average natural soil temperature (12°C), mycorrhiza did not improve plant nutrient capture but significantly enhanced plant P capture at 17°C. Photosynthetic nitrogen-use efficiency was higher at 17°C than at 12°C and was further increased by mycorrhiza at 17°C. Photosynthetic phosphorus-use efficiency was not affected by temperature or mycorrhiza. Increasing the growing temperature by 5°C increased the relative shoot growth rate by 15%. Mycorrhizal symbiosis did not enhance plant growth rate, but the plants gained between 20% and 90% more mycorrhiza-mediated P when grown at higher temperature. The results suggest that these low arctic species have good potential to respond positively to increasing temperatures.     

Endomycorrhizal and rhizobial symbiosis: How much do they share?

Abstract

Legume plants enter two important endosymbioses – with soil fungi, forming phosphorus acquiring arbuscular mycorrhiza (AM), and with nitrogen-fixing bacteria, leading to the formation of nitrogen-fixing root nodules. Both symbioses have been studied extensively because these symbioses have great potential for agricultural applications. Although 80% of all living land plants form AM, the nitrogen-fixing root nodule symbiosis with rhizobia is almost exclusively restricted to legumes. Despite varying degree of differences in the morphological responses induced by both endosymbionts in the host plants, significant similarities in the development of both fungal and bacterial symbioses have been reported. The signal perception and signal transduction cascades that initiate nodulation and mycorrhization in legumes partially overlap. Legume genes have been identified that are required for the establishment of both AM and root nodule symbiosis and are referred to as the common SYM genes. Genetic dissection of the common SYM signal transduction pathway required for bacterial and fungal root endosymbiosis has not only unraveled the players involved but also provided a first glimpse at conservation and specialization of signaling cascades essential for nodulation and mycorrhiza development. Based on the observation of common signaling cascades, it is tempting to speculate that the root nodule symbiosis, where fossil records date back to the late Cretaceaous, adopted and subsequently modified more ancient signal transduction pathways leading to AM formation, having already been in place 400 million years ago. This review discusses the common aspects of recognition of mycorrhizal fungi and Rhizobium by the host, and further signal transduction that leads to an effective symbiosis.     

Characterizing variation in mycorrhiza effect among diverse plant varieties

Exploitation of arbuscular mycorrhizal fungi may be an important approach for development of reduced-input agriculture. We discuss the use of linear models to analyze variation in mycorrhiza response among diverse plant varieties in order to assess the value of mycorrhizas. Our approach allows elimination of variation linked to differences in plant performance in the absence of mycorrhizas and the selection of plant lines that might harbor genetic variation of use to improve the mycorrhizal symbiosis in agriculture. We illustrate our approach by applying it to previously published and to novel data. We suggest that in dealing with a relative trait such as mycorrhiza effect, the choice of measure used to quantify the trait greatly affects interpretation. In the plant populations under consideration, we find evidence for a greater potential to increase mycorrhiza benefit than previously suggested.