Identification of the nitrogen-fixing Pseudomonas stutzeri major flagellar gene regulator FleQ and its role in biofilm formation and root colonization

Flagellar biosynthesis and motility are subject to a four-tiered transcriptional regulatory circuit in Pseudomonas,and the master regulator FleQ appears to be the highest-level regulator in this hierarchical regulatory cascade.Pseudomonas stutzeri A1501 is motile by a polar flagellum;however,the motility and regulatory mechanisms involved in this process are unknown.Here,we searched the A1501 genome for flagella and motility genes and found that approximately 50 genes,which were distributed in three non-contiguous chromosomal regions,contribute to the formation,regulation and function of the flagella.The non-polar mutation of fleQ impaired flagellar biosynthesis,motility and root colonization but enhanced biofilm formation.FleQ positively regulates the expression of flagellar class Ⅱ-Ⅳ genes,suggesting a regulatory cascade that is coordinated similar to that of the well-known P.aeruginosa.Based on our results,we propose that flagellar genes in P.stutzeri A1501 are regulated in a cascade regulated by FleQ and that flagellum-driven motility properties may be necessary for competitive rhizosphere colonization.

Exclosures restored the density and root colonization of arbuscular mycorrhizal fungi in Tigray, Northern Ethiopia

Introduction:Exclusion of grazing animals and tree plantations were among the methods used for the restoration of degraded lands in tropical semiarid areas.Exclosures can foster secondary forest succession by improving soil conditions and modifying microclimate for understory growth.This paper compared the arbuscular mycorrhizal fungi(AMF)spore density,root colonization of woody plants and soil chemical properties under exclosure with increasing age,and grazing land at different slope positions.Methods:The study was conducted in northern Ethiopia from 12 exclosure sites paired each with adjacent grazing land in total from 24 sites with four treatments replicated three times.In the entire study,216 plots were examined of which 108 were in exclosures and 108 in communal grazing lands.There were four age classes and three slope positions in each of the land uses.Composite soil and root samples were collected using nested plots measuring 100m2 from four sides of 1763 plants for spore enumeration and root colonization.Soils for chemical properties were collected from the four corners and center of 5 m×5 m plots.Results:All the 61 woody plant species that belong to 41 families were colonized by AMF.Spore density and root colonization were significantly higher in exclosures as compared to grazing land and increased with increasing ages of exclosures.Foot slope had significantly higher spore density and root colonization than middle and upper slopes.Soil chemical properties were significantly higher in the exclosure,oldest age of exclosure,and foot slope position(except P).AMF spore density and root colonization were significantly positively correlated with soil chemical properties.Conclusions:Exclosures are helpful to restore the AMF spore density and root colonization of woody species and soil fertility.

Colonization by Klebsiella variicola FH-1 stimulates soybean growth and alleviates the stress of Sclerotinia sclerotiorum

Sclerotinia stem rot,caused by Sclerotinia sclerotiorum,is a destructive soil-borne disease leading to huge yield loss.We previously reported that Klebsiella variicola FH-1 could degrade atrazine herbicides,and the vegetative growth of atrazine-sensitive crops(i.e.,soybean)was significantly increased in the FH-1-treated soil.Interestingly,we found that FH-1 could promote soybean growth and induce resistance to S.sclerotiorum.In our study,strain FH-1 could grow in a nitrogen-free environment,dissolve inorganic phosphorus and potassium,and produce indoleacetic acid and a siderophore.The results of pot experiments showed that K.variicola FH-1 promoted soybean plant development,substantially improving plant height,fresh weight,and root length,and induced resistance against S.sclerotiorum infection in soybean leaves.The area under the disease progression curve(AUDPC)for treatment with strain FH-1 was significantly lower than the control and was reduced by up to 42.2%within 48 h(P<0.001).Moreover,strain FH-1 rcovered the activities of catalase,superoxide dismutase,peroxidase,phenylalanine ammonia lyase,and polyphenol oxidase,which are involved in plant protection,and reduced malondialdehyde accumulation in the leaves.The mechanism of induction of resistance appeared to be primarily resulted from the enhancement of transcript levels of PR10,PR12,AOS,CHS,and PDF1.2 genes.The colonization of FH-1 on soybean root,determined using CLSM and SEM,revealed that FH-1 colonized soybean root surfaces,root hairs,and exodermis to form biofilms.In summary,K.variicola FH-1 exhibited the biological control potential by inducing resistance in soybean against S.sclerotiorum infection,providing new suggestions for green prevention and control.

Biodiversity of Arbuscular Mycorrhizal (AM) fungi in mangroves of Goa in West India

Seventeen mangrove species of eight families at seven river- ine and fringe habitats in Goa West India were surveyed for Arbuscular Mycorrhizal （AM） fungal diversity. Sixteen species were found to be mycorrhizal and one species showed no AM fungal colonization. AM root colonization was recorded at all seven sites and ranged from 6%-77%. Maximum root colonization was recorded in Excoecaria agal- locha （77%） and minimum colonization in Avicennia marina （6%）. Paris-type colonization was predominant at all sites. Auxiliary cells were recorded in roots of Acanthus ilicifolius, Ceriops tagal and Sonneretia alba. AM fungal root colonization and spore density varied by plant species and site. Site average spore density ranged from 1.84 spores.g^-1 to 0.54 spores.g-1 of soil. In total, 28 AM fungal species of five genera, viz. Glomus, Acaulospora, Scutellospora, Gigaspora and Entrophospora, were recovered. Glomus was the dominant genus, three species of which were sporocarpic forms. Maximum site species richness （SR） ranged from 16 to 5. Species richness was maximum in A. ilicifolius where seven species of three genera were recovered. Based on relative abun- dance （RA） and isolation frequency （IF）, two common species, viz. G. intraradices and A. laevis, were recovered from all seven sites.

Arbuscular mycorrhizal associations in different forest tree species of Hazarikhil forest of Chittagong, Bangladesh

Biodiversity of arbuscular mycorrhizal （AM） colonization and AM fungal spores were studied in the roots and rhizosphere soils of ,4cacia catechu （L.f）. Wild., A. mangium Willd, Anthocephala cadamba Miq., Artocarpus chaplasha Roxb., Chickrassia tabularis A. Juss., Swie- tenia macrophylla King., Tectona grandis L. from plantations; Albizia procera （Roxb.） Benth., ,4. falcataria L., Alstonia scholaris （L.） R. Br., ,4phanamixis polystachya （Wall.） Parker., Hydnocarpus kurzii （King.） Warb., Heynea trijuga Roxb., Lagerstroemia speciosa （L.） Pers., Messua ferrea Linn., Podocarpus nerifolia Don., Swintonia floribunda Griff., Syzygiumfruticosum （Roxb.） DC., S. grandis （Wt.） Wal. from forest and nursery seedlings of A. polystachya, A. chaplasha, Gmelina arborea Roxb. and S. cuminii （L.） Skeels from Hazarikhil forest, Chittagong of Bangladesh. Roots were stained in aniline blue and rhiz0sphere soils were assessed by wet sieving and decanting methods. The range of AM colonization varied significantly from 10%-73% in the plantations sam- ples. Maximum colonization was observed in A. mangium （73%） and minimum colonization was observed in C. tabularis （10%）. Vesicular colonization was recorded 15%-67% in five plantation tree species. The highest was in A. cadamba （67%） and the lowest was in T. grandis; A. chaplasha and C. tabularis showed no vesicular colonization. Arbuscular colonization was recorded 12%-60% in four plantation tree species. The highest was in .4. mangium （60%） and the lowest was in A. cadamba. Roots of Artocarpus chaplasha, C. tabularis and T. grandis showed no arbuscular colonization. Among 12 forest tree species, nine tree species showed AM colonization. The highest was in A. falcataria （62%） and the lowest was in S. fruticosum （10%）. Significant variation in vesicular colonization was recorded in seven forest tree species. The highest was in H. trijuga （52%） and the lowest was in L. speciosa 08%）. Hydnocarpus kurzii, M. ferrea, P. nerifolia S. fruticosum and S. grandis showed no vesicular colonization. Arbuscular colonization was recorded in sevenforest tree species. The highest was inA. falcataria （60%） and the lowest was in A. procera （10%）. All the nursery seedlings showed AM coloniza- tion and the range was 10%-73%. Vesicules were recorded in G. arbo- rea （40%） and S. cumini （40%）. Arbuscular colonization was recorded in G. arborea （100%） and S. cumini （100%）. Spore population was re- corded 77-432/100 g dry soils, 80-276/100 g dry soils, and 75-153/100g dry soils in plantation, forest and nurse.ry, respectively. Glomus and Acaulospora were dominant genera among the six AM fungi recorded. Significantly positive correlation was observed between/AM colonization and AM fungal spore population in Hazarikhil plantation tree species, forest tree species and nursery tree seedlings. The present study showed the biodiversity of root colonization and AM fungi are active in nutrient cycling, survivals and seedling establishment of the plants in the Hazarikhil forest, plantation and nursery.

Araucaria angustifolia Aboveground Roots Presented High Arbuscular Mycorrhizal Fungal Colonization and Diversity in the Brazilian Atlantic Forest

Almost 30 different arbuscular mycorrhizal fungi(AMF) species, distributed in different genera such as Glomus, Acaulospora,Scutellospora, Entrophospora, Ambispora, Kuklospora, Gigaspora, and Archeospora, have been identified in the root zone of Araucaria angustifolia, known as Brazil Pine. During our AMF survey in this ecosystem, our attention was called to the presence of many superficially growing Araucaria roots. Our hypothesis was that these roots were colonized with AMF because of the presence of AMF spores in organic material aboveground. Samples of these superficial roots and the organic substrate they were growing on were evaluated for their mycorrhizal status. DNA was extracted from the AMF-colonized superficial roots and submitted to polymerase chain reaction(PCR) amplification using the NS31-AM1 primer pair, followed by cloning and sequencing. We found that the root colonization percentages were between 31% and 52%, and the number of AMF spores in the substrate ranged from 27 to 164 spores per 50 g dry substrate. The phylogenetic analyses and tree construction using maximum parsimony(MP) and neighbor-joining(NJ)methods identified 13 different species of the phylum Glomeromycota belonging to the genera Glomus, Funneliformis, Rhizophagus,Gigaspora, Acaulospora, and Archaeospora, and five isolates were identified only at the genus level. To our knowledge, this is the first report on Araucaria angustifolia with roots growing aboveground, producing runner roots that develop on dead tree trunks and organic material. The higher colonization of the aboveground roots than those commonly found in belowground Araucaria roots suggests that they may present active metabolic uptake of nutrients.

A Preliminary Survey of Arbuscular Mycorrhizal Fungi Associated with Marsh Plants in Lhalu Wetland,Suburban Lhasa,South Tibet

A survey was made of the spore community of arbuscular mycorrhizal fungi (AMF) and root colonization by AMF in 16 plant species in Lhalu wetland on the outskirts of Lhasa city in Tibet. It was found that 13 of the 16 plant species investigated (81. 5% ) formed arbuscular mycorrhizal structures and dark septate endophytic fungi colonized the roots of most species. Total AMF colonization ranged from 0 to 82. 6% in dicots and 0 to 54. 5% in monocots. Both total AMF and arbuscular colonization were greater in dicots than that in monocots. A total of 48 taxa representing 7 genera of AMF were isolated and identified. Of these,9 species belonged to Acaulospora,2 to Appendicispora,34 to Glomus,and 1 each to Pacispora,Paraglomus and Scutellospora. Spores of Glomus aggregatum,G. deserticola and G. etunicatum were most common and abundant in the spore survey. Spores of 8 to 26 AMF species were isolated from the rhizosphere soil of individual plant species. Spore densities in soil associated with the 16 plant species ranged from 20 to 475 per 20 g soil,with an average of (92. 3 ± 14. 6). Species richness of AMF ranged from 6 to 12. 7. There were no significant differences between dicots and monocots in AMF spore density or species richness. Future work directed towards under- standing the response of the wetland plants to AMF may provide some insight into the role that these fungal symbionts may play in influencing plant diversity in this important urban wetland.

Arbuscular mycorrhizal fungi and endophytic fungi differentially modulate polyamines or proline of peach in response to soil flooding

Symbiotic fungi are involved in plant flooding tolerance,while the underlying mechanism is not yet known.Since polyamines(PAs)and proline are also associated with stress tolerance,it is hypothesized that the enhancement of stress resistance by symbiotic fungi is associated with changes in PAs and/or proline.The aim of this study was to analyze the effect of inoculation with Funneliformis mosseae and Serendipita indica on plant growth,PAs,and proline and the metabolisms in peach(Prunus persica)under flooding.Two-week flooding did not affect root colonization frequence of F.mosseae,while it promoted root colonization frequence of S.indica.Under flooding,plants inoculated with F.mosseae and S.indica maintained relatively higher growth rates than uninoculated plants.Funneliformis mosseae promoted root ornithine(Orn)contentration and arginine(Arg)and Orn decarboxylase activities under flooding,which promoted putrescine(Put),cadaverine(Cad),and spermidine(Spd)contentrations.Conversely,S.indica decreased contentrations of Arg,Orn,and agmatine and Arg decarboxylase activities,thus decreasing PA contentrations under flooding.Polyamines were negatively correlated with the expression of PA uptake transporter genes,PpPUT1 and PpPUT2,in peach.Polyamine transporter genes of F.mosseae(FmTPO)and S.indica(SiTPO)were regulated by flooding,of which FmTPO1 was positively correlated with Put,Cad,and Spd,along with positive correlations of Spd with SiTPO1,SiTPO2,and SiTPO4.Under flooding,F.mosseae decreased proline concentration,while S.indica increased proline concentration and correlated with expression of a△^(1)-pyrroline-5-carboxylate synthetase gene,PpP5CS2.It was thus concluded that F.mosseae modulated polyamine accumulation,while S.indica induced proline accumulation to tolerate flooding.

Screening of Streptomyces strains helping arbuscular mycorrhizal symbiosis against pepper(Capsicum annuum L.)Phytophthora blight

Mycorrhiza helper bacteria(MHB)can promote the formation and functioning of arbuscular mycorrhizal(AM)symbiosis,but their role and application potential in coping with soil-borne diseases are still unclear.A 14-week greenhouse pot experiment was conducted to obtain several actinomycete strains helping AM symbiosis in suppressing the Phytophthora blight of pepper(Capsicum annuum L.),using a soil inoculated with Phytophthora capsici after sterilization.Five Streptomyces strains,including S.pseudogriseolus,S.albogriseolus,S.griseoaurantiacus,S.tricolor,and S.tendae,as well as the AM fungus(Funneliformis caledonium)were tested.The Phytophthora blight severity reached 66%at full productive stage in the uninoculated control,and inoculation of F.caledonium,S.griseoaurantiacus,and S.tricolor alone significantly decreased(P<0.05)it to 47%,40%,and 35%,respectively.Compared to F.caledonium alone,additional inoculation of S.tricolor or S.tendae,which were isolated from the rhizosphere of a healthy individual in an infected field,significantly elevated(P<0.05)root mycorrhizal colonization,root biomass,fruit yield,and total K acquisitions of pepper and further significantly decreased(P<0.05)blight severity.According to the feature of enhancing disease-suppression by AM symbiosis,both S.tricolor and S.tendae were confirmed as MHB strains here.Specifically,S.tendae had a stronger performance in directly accelerating mycorrhization,while S.tricolor was also an antagonist to the pathogenic P.capsici.Furthermore,S.griseoaurantiacus with the independent disease-suppression function was not an MHB strain here.The redundancy analyses demonstrated that when AM fungus was present,root mycorrhizal colonization replaced soil pH becoming the main factor affecting pepper Phytophthora blight.Thus,S.tricolor and S.tendae seemed to have the value of preparation and application in the future to help AM symbiosis against pepper Phytophthora blight.

Responses of arbuscular mycorrhizal fungi to straw return and nitrogen fertilizer reduction in a rainfed maize field

Straw return can be used to reduce fertilizer input and improve agricultural sustainability and soil health.However,how straw return and reduced fertilizer application affect beneficial soil microbes,particularly arbuscular mycorrhizal fungi(AMF),remains poorly understood.Here,we conducted a five-year field experiment in a rainfed maize field on the Loess Plateau of northwestern China.We tested four treatments with straw return combined with four nitrogen(N)application rates,i.e.,100%,80%,60%,and 0%of the common N application rate(225 kg N ha^(-1)year^(-1))in this region,and two reference treatments(full or no N application),with three replicates for each treatment.Mycorrhizal colonization was quantified and AMF communities colonizing maize roots were characterized using Illumina sequencing.Forty virtual taxa(VTs)of AMF were identified in root samples,among which VT113(related to Rhizophagus fasciculatus)and VT156(related to Dominikia gansuensis)were the predominant taxa.Both root length colonization and AMF VT richness were sensitive to N fertilization,but not to straw return;furthermore,both gradually increased with decreasing N application rate.The VT composition of the AMF community was also affected by N fertilization,but not by straw return,and the community variation could be well explained by soil available N and phosphorus concentrations.Additionally,60%,80%,and full N fertilization produced similar maize yields.Thus,our study revealed the response patterns of AMF to straw return and N fertilizer reduction and showed that straw return combined with N fertilizer reduction may be a promising practice to maintain mycorrhizal symbiosis concomitantly with crop productivity.