植物根际促生菌作用机制研究进展

植物在生长过程中可能会遭受许多生物和非生物因素胁迫,从而降低生物产量. 人们已知一些植物在不同因素的刺激诱导下,能系统化建立抵抗或忍受不利因素的机制,植物根际促生菌（PGPR）就是其中一类能定殖于根系并促进植物生长的细菌.本文对PGPR促生机制进行归纳和总结,系统阐述了诱导体系抗性和诱导体系产生忍耐力两大促生机制.PGPR的作用机制的多样性暗示着其可能在更多的农业生态系统中得到应用.     

植物根际促生菌的筛选及鉴定

【目的】植物根际促生菌(PGPR)和植物的互作关系往往不稳定,PGPR菌群有可能提高菌株对野外环境的适应性。为此,本文根据PGPR促生机制的多样性,从不同植物根际土壤进行了PGPR的筛选及鉴定。【方法】首先,按照固氮、解磷、解钾、拮抗6种常见病原真菌,同时能在植物根际定殖为基本初筛标准,然后在实验室条件下测定初筛菌株的多项促生能力(PGP),最后通过生理生化试验和16SrRNA基因序列分析对所筛菌株进行鉴定。【结果】从江苏扬州、盐城等地土壤样品筛选出14株PGPR,具有体外抑菌、产NH3、产IAA、产HCN、产嗜铁素、解磷、溶钾、固氮以及产抗生素等促生能力。分类鉴定结果显示:7株属于假单胞菌属(Pseudomonas)、3株属于类芽孢杆菌属(Paenibacillus)、2株为芽孢杆菌属(Bacillus)、1株为布克霍尔德氏菌属(Burkholderia)、1株为欧文氏菌属(Erwinia)。【结论】所筛细菌具有多种促生能力,且能在根际定殖,为进一步构建多功能PGPR广适菌群提供菌株资源。     

Cytokinin production by fluorescent Pseudomonas in the presence of rice root exudates

Free-living bacteria may trigger the plant growth through production of phytohormones viz. gibberellins, auxins and cytokinins. A total 50 isolates of fluorescent Pseudomonas were screened for their ability to produce cytokinins such as isopentenyladenosine (IPA), dihydroxyzeatin riboside (DHZR) and zeatin riboside (ZR) as plant growth-promoting activity. Pseudomonas fluorescens AK1 and Pseudomonas aeruginosa AK2 were found higher phytohormones producing strains. Of the three cytokinins, IPA was the major cytokinin produced by both isolates in pure culture (5.5 and 2.9 pmol/ml, respectively) and with rice root exudates (5.9 and 3.4 pmol/ml, respectively). Production of ZR and DHZR for both organisms was found after 48 and 72 h. Amount of ZR and DHZR increased with time for both isolates in pure culture conditions. In presence of rice, production of ZR was increased 0.8 and 0.6 pmol/ml for P. fluorescence AK1 and P. aeruginosa AK2, respectively, in comparison with controls. There was no significant difference in the production of DHZR with rice exudates.     

Identification,characterization and optimization of phosphate solubilizing rhizobacteria (PSRB) from rice rhizosphere

Two billion people worldwide take rice (Oryza sativa L.) as a staple food. Phosphorus (P) and Nitrogen (N) are the major requirements of rice; although these are available in limited concentrations within rice growing regions. Among different types of Plant growth-promoting rhizobacteria (PGPR), Phosphate solubilizing rhizobacteria (PSRB) constitute an important class. These are known for plant growth promotion by enhancing P and N uptake. PSRB are nowadays used as biofertilizers to restore the soil health. Under the present investigation identification, characterization and optimization of phosphate solubilizing activity of these microbes at different pH, temperature and salt concentrations was carried out. Thirty-seven isolates were recovered from different regions of rice rhizosphere on Pikovskaya (PVK) agar among which 15 isolates were recovered from R.S. Pura, 12 isolates from Bishnah and 10 isolates were recovered from Akhnoor sector of Jammu, India. A prominent halo zone of clearance was developed around the colonies of 12 different isolates, indicating phosphate solubilization activity. Four distinct isolates were amplified, cloned and sequenced for taxonomic identification using 16S primers. The results indicated that PS 1, PS 2, PS 3, PS 4 were related to Pseudomonas aeruginosa, Bacillus subtilis strain 1, B. subtilis strain 2, B. subtilis strain 3, respectively. These strains when grown at a wide range of ecological factors showed maximum growth at pH between 6.8 and 8.8, temperature between 28 °C and 37 °C and salinity between 1% and 2%. Screening for phosphate solubilization activity revealed that the halo zone diameter formed by these isolates extended from 2.1 to 3.2 mm. The phosphate solubilizing efficiency (SE) ranged from 35.4 to 50.9 with highest value of 50.9 by PS4 and maximum P solubilization of 10.22 µg/ml was recorded by PS4 at 7th day. Phosphate solubilization activity of these identified PSRB strains can be utilized and explored in the rice growing belts of Jammu region which are deficient in phosphorus. MIC value for zinc sulphate heptahydrate in 12 isolates varied from 1 mg/ml to 6 mg/ml. Phosphate solubilization activity and MIC of these identified PSRB strains can be utilized and explored in the rice growing belts of Jammu region which are deficient in phosphorus.     

Evaluation of Ochrobactrum anthropi TRS-2 and its talc based formulation for enhancement of growth of tea plants and management of brown root rot disease

Aim: To evaluate Ochrobactrum anthropi TRS‐2 isolated from tea rhizosphere and its talc based formulation for growth promotion and management of brown root rot disease of tea. Methods and Results: Ochrobactrum anthropi TRS‐2, isolated from tea rhizosphere could solubilize phosphate, produce siderophore and IAA in vitro and also exhibited antifungal activity against six test pathogens. Application of an aqueous suspension of O. anthropi to the rhizosphere of nursery grown tea seedlings of five varieties of tea (TV‐18, T‐17, HV‐39, S‐449, UP‐3 and) led to enhanced growth of the treated plants, as evidenced by increase in height, in the number of shoots and number of leaves per shoot. Treatment with O. anthropi also decreased brown root rot of tea, caused by Phellinus noxius. Multifold increase in activities of chitinase, β‐1,3‐glucanase, peroxidase and phenylalanine ammonia lyase in tea plants was observed on application of O. anthropi to soil followed by inoculation with P. noxius. A concomitant increase in accumulation of phenolics was also obtained. Further, talc based formulation of O. anthropi was prepared and its survival determined every month up to a period of 12 months. Ochrobactrum anthropi could survive in the formulation up to a period of 9 months with a concentration of 7·0 log₁₀ CFU g^?1, after which there was a decline. Talc formulation was as effective as aqueous suspensions in both plant growth promotion and disease suppression. Conclusion: Ochrobactrum anthropi, either in aqueous suspension or as talc formulation induced growth of tea plants and suppressed brown root rot disease. It induced defense responses in tea plants. Significance and Impact of the Study: Ochrobactrum anthropi and its talc based formulation can be considered as an addition to available plant growth promoting rhizobacteria (PGPR) currently being used for field application. The present study offers a scope of utilizing this bacterium for growth promotion and disease management which would help in reduction of the use of chemicals in tea plantations.     

Induction of resistance against downy mildew on sunflower by rhizobacteria

Abstract

Induction of resistance to downy mildew caused by Plasmopara halstedii in sunflower was studied after treatment with PGPR (plant growth promoting rhizobacteria) strain INR7 (Bacillus spp). Treatment of sunflower seeds with 1×10⁸cfu/ml of PGPR strain INR7 resulted in decreased disease severity and offered 51 and 54% protection under green house and field conditions, respectively. The induction of resistance to P. halstedii by PGPR strain INR7 was accompanied by the accumulation of various host defense-related enzymes in susceptible sunflower seedlings. Enhanced activation of catalase (CAT), phenylalanine ammonia-lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO) and chitinase (CHI) was evident at 6, 9, 12, 12 and 12h post inoculation, respectively, in sunflower seedlings raised from seeds treated with PGPR strain INR7. This enhanced and early activation of defense-related responses in the susceptible cultivar after treatment with PGPR strain INR7 was comparable to that in the resistant cultivar. The results indicate that PGPR strain INR7 induced resistance against P. halstedii in sunflower is mediated through enhanced expression of defense mechanism.     

Biological hardening of micropropagated Picrorhiza kurrooa Royel ex Benth., an endangered species of medical importance

Three plant growth-promoting rhizobacteria viz. Bacillus megaterium, B. subtilis and Pseudomonas corrugata were used for biological hardening of micropropagated plantlets of Picrorhiza kurrooa. The bacterial isolates antagonized the fungal spp. postulated to cause death of micropropagated plants in plate-based assays and positively influenced survival and growth parameters in greenhouse investigation.     

植物根际促生菌对3种土传真菌病害病原的抑制作用

【目的】获取促生同时可防治3种土传真菌病害(Fusarium oxysporum、Sclerotinia sclerotiorum和Rhizoctonia solani)的生防菌,并明确其抑菌效果。【方法】利用前期研究获得的17株促生菌,采用平板对峙法测定其对病原真菌的拮抗作用及对菌丝生长的抑制作用。【结果】可有效拮抗立枯丝核菌的生防菌有6株,其中促生菌株FX2和LM4-3的抑制率达73.82%;拮抗尖孢镰刀菌的生防菌有7株,其中FX2的抑制率达到66.81%;拮抗油菜菌核病菌的生防菌有4株,其中菌株LHS11的抑制率高达85.71%。菌株LHS11和JM170通过次生代谢物抑制病原真菌。所有的生防菌对病原菌的菌丝生长均有一定的抑制作用。【结论】筛选得到对3种真菌病害病原具有较好生防作用的菌株LHS11和FX2。     

The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces resistance in tomato with induction and priming of defence response

In a previous study, we demonstrated the ability of the rhizobacterium Bacillus cereus AR156 (AR156) to protect tomato against bacterial wilt caused by Ralstonia solanacearum and root-knot disease caused by Meloidogyne incognita. Here, we investigate the ability of AR156 to promote plant growth and its role in the systemic protection of tomatoes cultivated in greenhouses against bacterial speck disease caused by Pseudomonas syringae pv. tomato DC3000 (DC3000). In our experiments, the AR156 population reached 10⁵–10⁶ CFU/g rhizosphere soil, and remained at that level in the rhizosphere of tomato plants for more than 2 months. In terms of its ability to promote plant growth, AR156 increased the average biomass of the tomato by 47.7%. AR156 also elicited induced systemic resistance against DC3000, significantly reduced bacterial speck disease severity 1.6-fold, and inhibited proliferation of the pathogen by approximately 15-fold. This strain triggered the accumulation of defence-related genes (PR1 and PIN2) in tomato leaves and primed the leaves for accelerated defence-related gene expression upon challenge with DC3000. That suggested simultaneous activation of the salicylic acid and the jasmonic acid dependent signalling pathways by AR156 against DC3000. In conclusion, B. cereus AR156 was found to form robust colonies in the roots of tomato and had some beneficial effects, including biological control of bacterial speck disease via ISR and promotion of plant growth.     

高产吲哚乙酸及高泌氨巴西固氨螺菌的筛选与鉴定

目的：从玉米根际和土壤中分离具有高产吲哚乙酸较强的泌氨能力的巴西固氮螺菌。方法：分别通过半固体NFb培养基、CR培养基、LB培养基分离培养固氮菌株,并经过一系列菌落菌体形态特征、生理生化特性和16S rDNA序列测定等试验对其进行鉴定。结果：经分离纯化获得10株固氮菌,并鉴定均为巴西固氮螺菌（Azospirillum brasilense）,其中菌株R7在甘油半固体培养基上能分泌约14mmol/L的氨,在添加了色氨酸的培养基中能够合成58.8μg/ml的吲哚-3-乙酸（IAA）。结论：成功筛选得到一株既高产吲哚乙酸又有较强的泌氨能力的巴西固氮螺菌。     

Diversity of rhizospheric and endophytic bacteria isolated from dried fruit of Ficus carica

There is currently an increasing demand for the characterization of endophytic bacteria isolated from different parts of plants (rhizosphere, roots, fruit, leaf) in order to improve the organic agriculture practices. The current research was performed to identify both rhizospheric bacteria isolated from the rhizosphere of Ficus carica in three different sites in the north of Tunisia and endophytic bacteria isolated from dried figs. We then characterized them for a diversity of plant growth-promoting (PGP) activities. A collection of 120 isolates from rhizospheric soil and 9 isolates from dried figs was obtained and purified. 16SrDNA gene amplification of rhizospheric bacteria revealed significant diversity and allowed for the assigning of the isolates to 6 phyla: Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Representative strains of the collection (90 strains) were tested for numerous PGP activities and resistance to abiotic stresses. The most common PGP trait for all bacteria from the three regions was siderophore production (62%), followed by cellulase (38%), then protease activity (37%), then by lipases activity (17%) and lastly by solubilization of phosphates (9%). Twenty -three strains that showed most PGP traits were selected, 8 strains presented 12 or more, and 15 strains displayed between 7 and 11 of 17 PGP activities. The majority of the isolates manifested a possible adaptation to abiotic stress and unfavorable environments. PCR-DGGE analysis of soil rhizosphere of the three sites allowed also for the acquisition of a Cluster analysis of rhizospheric bacterial communities. Our current study identified and characterized for the first time in Tunisia rhizospheric and endophytic bacteria from dried fruit of Ficus carica.     

强化龙葵富集镉根际促生菌的分离、筛选与鉴定

【背景】植物-微生物联合修复土壤重金属污染日渐兴起,获取与超富集植物高效互作的微生物是实现联合修复技术进步的关键。龙葵在镉农田污染修复中广泛应用。【目的】筛选可促进龙葵生长及富集镉的耐镉根际促生菌。【方法】从龙葵根际土分离耐镉菌株,筛选具有良好促生特性的菌株,水培试验考察镉胁迫下菌株对植物生长及镉富集能力的影响,确定可促进龙葵生长及富集镉的微生物菌株,通过生理生化特性和16S rRNA基因序列分析进行菌株的初步鉴定。【结果】分离得到NT1、AXY1、AW2和AW1四株强化龙葵富集镉促生菌,经鉴定分别为Lysinibacillus sp.、Beijerinckia fluminensis、Achromobacter animicus和Herbaspirillum huttiense。上述菌株均可有效促进龙葵生长,增加其株高和干物质积累,提高地上部镉富集量。其中,NT1可使株高、地上部干重分别增加31.33%和62.65%,AW2可使地上部镉富集量增加37.29%。【结论】筛选所得菌株可为提高植物修复效率提供实践依据,为研制田间施用生态功能菌剂做铺垫,用于农田镉污染的微生物-龙葵联合原位修复。     

Biological control of the wheat root rot caused by Fusarium graminearum using some PGPR strains in Saudi Arabia

The aim of this study was to evaluate the efficacy of selected bacterial strains against the wheat soil‐borne pathogen Fusarium graminearum under greenhouse conditions. The most potent isolates were 3 isolates out of 18 isolates, which have numbers 3, 9 and 10 with in vitro inhibition index 42.5%, 41.3% and 46.3% respectively. Isolates 3 and 10 were selected for the following experiments. Isolates 3 and 10 were identified as Bacillus subtilis MAA03 and Pseudomonas fluorescens MAA10, respectively according to International Identification Keys and, confirmed by using Biolog system and 16S rDNA where the strains exhibited more than 99.5% sequence identity. Their close taxonomic relationship was further documented by phenotypic similarities. The using of B. subtilis and P. fluorescens separately or in mixture as biocontrol agent against F. graminearum on wheat significantly increased the final germination percent, the mean daily germination and germination index of wheat cultivar, while the mean germination time was significantly decreased relative to infested control. The final infection percent, the mean daily infection and infection index were decreased significantly, while the mean infection time was significantly increased relative to infested control. The use of P. fluorescens as biocontrol agent was the most efficient than B. subtilis or in mixture and the best treatment was seed coating. The application of B. subtilis and P. fluorescens separately or in combination significantly affected the growth parameters of wheat cultivar Tabuki, the root length was significantly increased in seed coating and seed soaking treatments, while non‐significantly decreased in case of soil drench treatment relative to infested control. Shoot length was significantly decreased in case of seed coating treatment relative to infested control. The shoot fresh and dry weights were significantly increased in seed coating and seed soaking treatments relative to infested control. The root fresh and dry weights were significantly increased in seed coating and seed soaking treatments relative to infested control. The number of leaves was significantly increased in all treatments relative to infested control.     

Assessment of SCAR markers to design real‐time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize

Aims: To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real‐time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP‐154 and CFN‐535 in the rhizosphere. Methods and Results: Primers were designed based on strain‐specific SCAR markers and were screened for successful amplification of target strain and absence of cross‐reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real‐time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 × 10³ (for UAP‐154) and 4 × 10⁴ CFU g^?1 (for CFN‐535) in the maize rhizosphere. Inoculant quantification was effective from 10⁴ to 10⁸ CFU g^?1 soil. Conclusion: BOX‐based SCAR markers were useful to find primers for strain‐specific real‐time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. Significance and Impact of the Study: Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation‐independent monitoring methods were lacking. The real‐time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP‐154 and CFN‐535.     

Pseudomonas fluorescens enhances resistance and natural enemy population in rice plants against leaffolder pest

Plant growth promoting fluorescent pseudomonad strains Pf1, TDK1 and PY15 were evaluated for their efficacy against leaffolder ( Cnaphalocrocis medinalis ) pest in rice plants under field conditions individually and in combinations. Application of mixtures of Pseudomonas fluorescens strains Pf1, TDK1 and PY15 significantly reduced the leaffolder damage in rice plants compared with untreated control. Interestingly, natural enemy population in plots treated with P. fluorescens was greater than the chemical and untreated controls. Further, support for these results was gathered by assaying activities of polyphenol oxidase (PPO) and lipoxygenase (LOX) under glasshouse conditions. The results showed the higher activity of PPO and LOX in plants treated with P. fluorescens mixtures (Pf1 + TDK1 + PY15) than the plants treated with individual strains, chemical and untreated controls. Further, fluorescent pseudomonad mixtures increased the rice yield compared with individual strain and non-bacterized treatments. The present study reveals that in addition to plant growth promotion, plant growth-promoting rhizobacterial (PGPR) strains-mediated induction of PPO and LOX in rice plants could be involved in enhanced natural enemy populations and resistance mechanisms against leaffolder attack.     

Integration of plant growth-promoting rhizobacteria and chemical elicitors for induction of systemic resistance in mulberry against multiple diseases

Abstract

In mulberry (Morus alba L.), various individual strains of plant growth-promoting rhizobacteria (PGPR) and synthetic analogs of naturally occurring plant activators have demonstrated their potential to elicit induced systemic resistance (ISR) against either brown leaf spot (Cercospora moricola) or leaf rust (Cerotelium fici) diseases. However, these biological and chemical elicitors have not been evaluated so far against multiple infections of both these diseases which commonly occur during the post-rainy season. The present study was therefore aimed to assess the capability of PGPR strains and chemical plant activators, as individual and in integration, in elicitation of ISR against multiple infections. Three PGPR strains, Azotobacter chroococcum strain Azc-3, Bacillus megaterium strain Bm-1 and Pseudomonas fluorescens strain Psf-4, and plant activators, acetyl-salicylic acid (ASA), sodium salicylate (NaS) and 4-amino-n-butyric acid (ABA) were selected for the study. Under in vitro tests, all the plant activators up to 2000 ppm concentration exhibited their compatibility with the PGPR strains tested. Upon assaying of elicitors with plant-pathosystem, disease suppression was significantly (p = 0.05) high with integrated application of PGPR strains and plant activators when compared to their individual applications. All the elicitors at individual application varied in their response to multiple infections with the plant age. However, integration of Azc-3 + ASA provided greater suppression to multiple infections of brown leaf spot and leaf rust diseases during the entire growth period of mulberry plants. Thus, this combination of biological and chemical elicitors holds great promise to provide an effective ecofriendly alternative to the toxic chemical fungicides presently recommended for the control of brown leaf spot and leaf rust diseases in mulberry.