Interaction of Kreyellidae sp. and plant growth promoting bacteria influences the soil characteristics and root structure of rice plants

doi:10.21203/rs.3.rs-924357/v1

Download PDF

Research Article

Interaction of Kreyellidae sp. and plant growth promoting bacteria influences the soil characteristics and root structure of rice plants

https://doi.org/10.21203/rs.3.rs-924357/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Plant growth and productivity depend on the complex and dynamic interaction between the plant roots and soil microflora. At present, the research on rhizosphere associated microbes’ is largely focused on bacteria and fungi; whereas the interaction of soil protists with plants and other microbes remain unexplored. The present study aimed to investigate the impact of a ciliate (Kreyellidae sp. C5) and two plant growth-promoting bacteria (PGPB) i.e., Pseudomonas sp. (Ps) and Enterobacter sp. (Ec), on the growth of rice plants. It was observed that the protist-PGPB interaction significantly modified the root structure leading to an enhanced outgrowth of lateral roots (272.08% − 380.41%) and seminal roots (190.40% -250.45%), in addition to an increase in the primary root length (Turkey’s HSD, p < 0.05). The Phospholipid Fatty Acid (PLFA) analysis indicated a striking shift in the overall soil microbial communities due to the presence of a predator. The combined treatments (with C5Ps and C5Ec) further increased the Microbial Carbon Biomass (MBC) to 223.59% and 310.57% as compared to control and PGPB treatments respectively. A similar enhancement of dehydrogenase enzyme activity was observed in soil samples of rice plants on combined treatments. In contrast, the alkaline phosphatase and fluorescein diacetate enzyme activities were recorded to be more in soil samples treated with PGPB. The combined treatment of rice plants also enhanced the uptake of N and P moderately, as compared to PGPB treated plants. However, this enhancement was significant compared to control plants. The colony-forming unit (CFU) and most probable number (MPN) was found to be more in C5Ec (131.0 ± 3.70×10¹¹ and 5.12 ± 0.06) and C5Ps (24.10 ± 2.19×10¹⁰ and 10.52 ± 0.39), as compared to control and PGPB treated soil samples. In conclusion, this is the first study that demonstrates significant modification of root structure and increased nutrient uptake by rice plants through interaction between Kreyellidae sp. and PGPB. In addition, we also report improved respiration and diverse microbial population in soil samples on combined treatment of rice plants.

Agroecology

Evolutionary Biology

Ciliates

Kreyellidae

PGPB

PLFA

Predator

Soil respiration

The rhizosphere is the region of soil surrounding the plant roots. It is densely populated with life forms of different domains (insects and micro-organisms), and hence considered as a hotspot for microbial interactions [1, 2]. In the soil ecosystem, plant roots provide nourishment to soil microbes by releasing root exudates. In turn, the Plant Growth-Promoting Bacteria (PGPB) in the surrounding soil stimulates plant growth through direct and/or indirect mechanisms. Direct mechanisms include nitrogen fixation, solubilization of macro- and micro-nutrients from the soil, production and/or regulation of phytohormones. The indirect mechanisms involve suppression of phytopathogens and induced systemic resistance in plants against a broad range of phytopathogens [3]. The PGPB are extremely valuable for sustainable agricultural processes. However, their survival and efficacy in soil are the most important factors for sustainable plant growth promotion.

It has been reported that the presence of protists in the rhizosphere region increases the activity and survival of PGPB in soil [4]. It is observed that selective grazing by protists induces shifts in the microbial communities, which favours the growth of indole-3-acetic acid (IAA) producing bacteria [5] or nitrifying bacteria [6]. Since IAA is responsible for the development of lateral root primordia and root elongation in plants, it favors plant growth promotion via enhanced nutrient uptake through increased root surface area. The stimulated nitrogenase activity of Stenotrophomonas sp. and Azospirillum lipoferum in response to ciliate predation pressure has also been reported recently [7]. In addition, the presence of protists also stimulates the biocontrol activities of certain bacteria against soil-borne plant pathogens. The increased production of siderophores [8, 9], up-regulation of cyclic lipopeptides [10] and 2, 4-DAPG production [11] by Pseudomonas fluorescens has also been suggested earlier in the presence of amoebae. In another study, production of secondary metabolites by P. fluorescens in response to grazing improved its competitive advantage over the other bacteria [12], which proved to be beneficial for plant growth and overall health. Even when grazed upon by amoebae, it was found that 2, 4-DAPG synthesis was upregulated by P. fluorescens [11]. This compound directly induces systemic resistance in plants [13]. In literature, most reports on the effect of protist-bacteria interaction on plant growth promotion have used amoebae as a model predatory species. The involvement of ciliate species in plant growth promotion studies is rare, except for some recent reports [14–16]. The capability of ciliate to feed on a wide variety of bacterial species depending upon size, motility, pigmentation and microcolony formation [17] has been well demonstrated in literature. However, the impact of ciliate predation on PGPB and their combined influence on plant growth has been an under-researched topic.

Therefore, the present investigation was carried out to study the impact of ciliate species (having a predation effect on PGPB) on plant attributes. For this purpose, the ciliate species was isolated from rice rhizosphere soil and two previously characterized PGPB i.e., Pseudomonas sp, and Enterobacter sp. [18, 19] were used to study their effects on rice plants. Further, the impact of bacterial and ciliate interactions on soil respiration, nutrient uptake by plants and phospholipid profiles of soil samples were also studied.

Microorganisms and culture conditions

Two previously characterized PGPB, i.e., Pseudomonas sp. and Enterobacter sp., were selected for the present study [18, 19]. Both the cultures were pre-grown in nutrient broth before performing each experiment.

A ciliate strain designated as C5 was isolated from rice rhizosphere soil sample by following a standard protocol [20]. The ciliate was separated from the soil slurry using micro capillary tubes and transferred to sterile Page’s saline on a microscopic slide. After confirming the single organism under the microscope, the ciliate was transferred to Nutrient broth (1 %) in Neff’s Modified Amoebae Saline (NB-NMAS) medium containing heat-killed suspension of E. coli as a food source. For identification, proteinase K digestion method was followed with some modifications [20] to extract the genomic DNA. The extracted DNA was used as template for 18S rDNA amplification using eukaryotic specific primers EU347F (5’-AGG GTT CGA TTC CGG AGA-3’) and EU929R (5’-TTG GCA AAT GCT TTC GC-3’) [21]. PCR reaction was carried out in 50 µl volume reaction mixture containing 10X buffer with MgCl₂ (5 µl), 0.25 mM dNTP mixture (5 µl), 1U Taq DNA polymerase (0.7 µl), 0.1 µM forward (2.5 µl) and reverse primer (2.5 µl) and 5 µl template DNA extracted from ciliate. The thermal cycler was programmed as follows: initial incubation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 45 sec, annealing at 53°C for 1 min, and extension at 72°C for 1 min, followed by final extension step of 10 min at 72°C. The sequencing of PCR products was carried out at National Collection of Industrial Microbes, Pune, India. The obtained 18S rDNA sequence was compared with 18S rDNA gene sequence available at NCBI GenBank database (http://www.ncbi.nlm.nih.gov) using BLASTn programme for identification of the ciliate species.

Growth Assessment Assay Of Ciliate Species With Pgpb

The fresh cultures of selected PGPB were streaked (two lines) across the 0.2 X nutrient yeast extract agar plates [22]. After 3 h of incubation, 5.0 µl (200 cysts µl^− 1) of ciliate were spot inoculated on one end of the streaked line. The other streaked line (of PGPB) was used as a control to compare the changes in the growth pattern. The inoculated plates were incubated at Room temperature (RT) for 5 days under dark conditions. On the other hand, the ciliate species (20,000–25,000 cysts ml^− 1) in the broth medium were added to a 20 ml culture suspension flask and allowed to acclimatize for 3 h before the introduction of PGPB (1.1×10⁷ to 2.26×10⁷ CFUs ml^− 1). The flasks were incubated at RT on a rotary shaker at 50 rpm for up to 5 days. MPN and CFU counting was done using 1 ml aliquots from each flask, for 5 days, using Neubauer’s chamber under the light microscope and on nutrient agar plate respectively. Each experiment was performed in triplicates.

Impact of ciliate-PGPB interaction on plant growth

Plate assay

The effect of ciliate-PGPB interaction on the growth of rice plant was studied using plate assay method [23]. For this purpose, rice seeds (Gurjari variety) were surface sterilized in 70% ethanol for 5 min, followed by treatment with 2% v/v sodium hypochlorite for 10 min. After 8–10 washes with sterile distilled water, the seeds were transferred to a 24-well microtiter plate containing 100 µl sterile distilled water and incubated at 20°C in the dark until germination. After 3 days, the germinated seeds were fixed on the edge of the surface of NB-NMAS agar plates [23]. This media was selected for our study since it avoids the overgrowth of bacteria (due to low concentration of nutrients) and supports the growth of protists species. To prepare the agar plates, 10 ml NB-NMAS medium was poured in sterile Petri dishes (110 × 15 mm, diameter × height) and kept at an angle of 30° until solidification. The experiments were set up for three different treatments; 1) control (without bacteria or ciliate inoculation), 2) only PGPB inoculation, and 3) PGPB plus ciliate inoculation. The interactive effect of ciliate-PGPB on early rice plant growth was determined by measuring the shoot length, primary root length and number of seminal and lateral roots of rice seedlings after 10 days of incubation.

Pot Assay

For the pot assay, the experiments were set up in plastic pots (12 inches) under greenhouse conditions. The pot experiments were conducted using loam soil collected from the agricultural fields (pH 7.2; N₂- 7.3%; organic carbon-1.28%; P-403.2 mg/kg; K-288.7 mg/kg). The surface-sterilized rice seeds (as mentioned above) were exposed to five different treatments and subsequently shade dried for 2 h before sowing into the pots. The treatments were done using 24 h old bacterial culture (10⁸ CFUs ml^-) and active ciliate culture (10³ ml^-). The experimental setup included (1) control (without any treatment), (2) Enterobacter sp. (Ec) alone (3) Pseudomonas sp. (Ps) alone, (4) Enterobacter sp. + C5 (C5Ec) and (5) Pseudomonas sp. + C5 (C5Ps). Five replicates were used for each treatment in a completely randomized block design. The pots were watered every alternate day to retain moisture content, and they were maintained at natural day and night conditions in the greenhouse. After one month, the plants were harvested to measure root length, shoot length and dry weight.

Analysis Of Soil Samples

The bacterial communities in the soil samples were enumerated as colony-forming unit (CFU) g^-1 while protists were enumerated using modified Singh’s most probable number (MPN) technique [24]. Total N and P content in the plant biomass were determined based on the methods described by Gebremikael [25] and Hogue [26] respectively. The Phospholipid Fatty Acid Analysis (PLFA) was undertaken at Royal Life Sciences Pvt. Ltd, Secunderabad, India [27]. The soil respiration analysis was conducted using fluorescein diacetate (FDA) hydrolysis assay of the soil samples using the method described by Adam and Duncan [28]. Dehydrogenase activity in soil was estimated using a 2-3-5-triphenyl tetrazolium chloride (TTC) reduction method [29]. The alkaline phosphatase activity of the soil samples was estimated by the method described by Bessey [30]. The microbial carbon biomass was estimated by the chloroform fumigation incubation method described by Anderson and Ingram [31]. The microbial biomass C (MBC) was calculated using the following equations

$$\text{O}\text{r}\text{g}\text{a}\text{n}\text{i}\text{c} \text{C} \left(\text{%}\right)= \frac{\left(\text{A} \times \text{M} \times 0.003\right)}{g}x \frac{\text{E}}{S} x 100$$

$$\text{A}=\left{ml \left(HB\right)- \text{m}\text{l} \left(\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\right)\right}\text{x} \frac{\text{m}\text{l} \left(\text{U}\text{B}\right)- \text{m}\text{l}\left(\text{H}\text{B}\right)}{\text{m}\text{l} \left(\text{U}\text{B}\right)}+\left{\text{m}\text{l}\right(\text{H}\text{B}) – \text{m}\text{l}(\text{H}\text{B}) – \text{m}\text{l}(\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\left)\right}$$

$$\text{M}\text{B}\text{C} =\left{\text{O}\text{r}\text{g}\text{a}\text{n}\text{i}\text{c} \text{C} \right(\text{f}\text{u}\text{m}\text{i}\text{g}\text{a}\text{t}\text{e}\text{d} \text{s}\text{o}\text{i}\text{l})- \text{O}\text{r}\text{g}\text{a}\text{n}\text{i}\text{c} \text{C} (\text{U}\text{n}\text{f}\text{u}\text{m}\text{i}\text{g}\text{a}\text{t}\text{e}\text{d} \text{s}\text{o}\text{i}\text{l}\left)\right} \text{x}2.46$$

where, M = Molarity of ferrous ammonium sulphate (0.033M); G = Dry soil mass (g); E = Extraction volume (ml); S = Digest sample volume (ml); HB = Heated blank and UB = Unheated blank.

Statistical analysis

Statistical analysis of plant growth parameter, soil respiration and nutrient uptake were analyzed using one-way analysis of variance (ANOVA) in SPSS version 23.0. Comparisons between means were performed at a 5% probability level with a Tukey-test (Tukey’s HSD). Principal component analysis was carried out using Minitab software to differentiate the PLFAs of the soil microbial community.

Identification of the ciliate

The isolated ciliate was identified based on 18S rDNA sequencing technique. The results revealed that the strain C5 belonged to the genus Kreyellidae (MW274681).

Growth assessment assay with PGPB

The growth assessment assay of predator-prey interaction revealed that Kreyellidae sp. suppressed PGPB (Enterobacter sp. and Pseudomonas sp.) growth on NYEA (Fig. 1A). This was ascertained by visual observations under the light microscope (10X) and agar plates showing confluent growth elimination along the streaked line of test bacteria. However, in the liquid medium, the bacterial response to predation pressure was different. Enterobacter sp. count reduced sharply from 1.98 × 10⁹ to 3.4 × 10⁵ CFUs ml^-1 under ciliate predation pressure after 24 h, whereas Pseudomonas sp. count remained constant at 2.57 × 10¹¹CFUs ml^-1 for 96 h (Fig. 1B). The drastic increase in a logarithmic growth state of ciliate, in response to bacteria, was observed from 24 to 72 h. After 72 h, the growth rate reduced, and with time, the ciliate was encysted. Though the growth and encystment in response to Pseudomonas sp. and Enterobacter sp. were different, neither of the bacterial species harmed the ciliate (Fig. 1C).

Interactive effect on plant growth

The study of combined impact of both the organism on plant growth revealed more ramified root structure and increased shoot length in plate assay (Table 1). The combination of ciliate and PGPB (C5Ps and C5Ec) significantly enhanced the formation of lateral roots (272.08% and 380.41%) and seminal roots (190.40% and 250.45%). The combined treatment also increased shoot and primary root length as compared to control and bacterial treatment alone (Turkey’s HSD, P<0.05). A similar effect was also observed with pot studies (Table 2). A significant variation was observed in the root length on combined treatment of seeds (Fig. 2; Turkey’s HSD, P<0.05). Overall, it was observed that the combined treatments led to the development of significantly different root structure. There was not much variation observed in the shoot length on any of the treatments in this study. However, significant enhancement in dry weight of shoot and roots of rice plant was observed on treatment with C5Ps and C5Ec. Our results indicated that Ec significantly increases the plant growth compared to control; however, the combination of C5 + Ec had the highest impact on lateral root formation and primary root length.

Densities of bacteria and protists in soil

Enumeration of bacteria and protist from the soil sample revealed that the total number of cultivable bacteria varied depending upon the treatment (Turkey’s HSD P<0.05). The CFU was found to be greater in C5Ps (24.10±2.19×10¹⁰) and C5Ec (131.0±3.70×10¹¹) treatment compared to control and PGPB treatment alone (Table 3). In case of the MPN study, the protist population was found to be higher on C5Ps (10.52±0.39) and C5Ec (5.12±0.06) treatment (Table 3).

Shifts in microbial community composition

Principal component analysis (PCA) was applied to the PLFA data, with principal components of 1 (PC1) and 2 (PC2) accounting for 75.6% of total variation in microbial community composition (Fig. 3). The PCA plot revealed that the soil microbial communities observed after different treatments were compositionally distinct. The variation in the PCA separated the combined treatments and PGPB treatments from control (Fig. 3A). Although the PLFAs of C5Ec and C5Ps were different, the PLFA of Ps and C5Ec were closely related. Notably, as indicated in the PCA of all treatment scenarios, the Gram-positive, Gram-negative and fungal communities were found to be higher in control samples as compared to treated samples, whereas PLFAs of eukaryotes were found to be strikingly higher in all treated samples except in the control treatment. The relative abundance of arbuscular mycorrhizal fungi, methanotrophs and actinomycetes were negatively correlated in control samples as compared to treated samples (Fig. 3B).

Microbial carbon biomass and soil respiration activities

A significant difference was observed in the MBC (Table 4) in the presence of ciliate and PGPB compared to control. The treatment with C5Ps increased the MBC to 223.59% (control) and 8.90% (Ps treatment) while C5Ec treatment increased the MBC to 310.57% (control) and 33.46% (Ec treatment). A similar activity was observed for the soil dehydrogenase enzyme. However, the alkaline phosphatase and FDA enzyme activities were greater in PGPB treated samples as compared to combined treatments. The alkaline phosphatase activity on different treatments was observed in the following sequence; Ps>C5Ps>Ec>C5Ec>control whereas for FDA activity it was noted as Ps>control>Ec>C5Ps>C5Ec (Table 4).

Nitrogen and phosphorus content of rice plant

The inoculation of ciliate-PGPB combination (C5Ps and C5Ec) moderately enhanced N and P uptake in the plant samples. The PGPB and ciliate treatments alone also showed a positive impact on total N and P (Table 5). Although this impact was much less compared to combined treatment, it was relatively significant compared to control plants.

The interactions between a soil protist and PGPB have been demonstrated in the present study where Enterobacter sp. density negatively correlated with ciliate density, whereas Pseudomonas sp. proved to be resistant to predation under laboratory conditions. The results of this study are in agreement with Pedersen [32] and Amacker [33], where Pseudomonas sp. was reportedly unaffected by ciliate predation. This may be due to the production of specific anti-predatory secondary metabolites by Pseudomonas sp. [22]. On the other hand, Enterobacter sp. are not known to produce toxic compounds. During pot culture studies, enhanced survival of both PGPB cultures was observed against predation. A possible explanation for this observation is the selective feeding of the introduced ciliate on competitors in the soil. This would have decreased the nutrient competition for Enterobacter sp. and hence aided in its survival [34].

Although a positive impact of rhizosphere protists (especially Acanthamoeba sp.) on plant productivity has been described repeatedly in literature; very few reports are available, at present, on the impact of protists on the survival of PGPB [5, 34]. For instance, the treatment of rice seedlings with amoebae developed a more ramified root structure and increased N uptake by the plants [23]. Similarly, our observations are in agreement with well-documented studies on the effect of soil protists on rice [23, 35] and other plants [5, 36, 37]. This observation may be due to the well-known microbial loop concept which explains the nutrient turn-over by protists preying on bacterial community and subsequently releasing bacterial biomass [38]. Therefore, the plants can take up considerable amount of readily available N due to the protist predation on combined treatments.

In our study, the highest increase in plant growth was obtained on combined treatment with Kreyellidae sp. and Enterobacter sp. It is known that the production of phytohormone by bacteria stimulates the plant growth [39] and recent reports have suggested that the predation by protist increases the IAA producing bacterial community in soil and thereby stimulates the early rice plant growth [34]. The current results of increased plant growth and higher densities of bacteria on combined treatment with Kreyellidae sp. and Enterobacter sp. further explains the positive impact of ciliate-PGPB interaction on plant growth performance. However, different protist and PGPB interaction should be studied to better understand the impact of their interaction on plant growth.

It has been reported in the previous studies that protist inoculation changes the soil microbial community composition [40, 41]. Our results are in agreement with these findings of top-down regulation by protists in structuring the soil microbiota. Different protist species are fundamentally differed in their grazing preferences [42]. For instance, inoculation of Tetrahymena increased the Gram-positive PLFAs, whereas, Colpoda led to an increase in Gram-negative PLFAs [15]. In our studies, Kreyellidae sp. decreased the PLFAs of Gram-positive as well as Gram-negative bacteria. The substantial decrease in Gram-positive bacterial community in combined and PGPB treatments was likely due to the competitive shifts in the bacterial community and preferential predation of ciliate [12, 43]. Our results suggested fundamentally different effects of ciliate grazer on overall soil microbial community in rice rhizosphere.

The MBC is an efficient parameter in evaluating the plant health since it involves determining the regulated nutrient cycles which is made available to plants [44]. In the present study, MBC was greater in microbial treated plants as compared to controls. This indicated that the ciliate-PGPB interaction could stimulate the microbial activity in soil. Similarly, the increased dehydrogenase activity on combined treatment might be due to the presence of vast quantities of readily degradable organic substrates for microbial proliferation [44]. The difference in the alkaline phosphatase and FDA activities of the treated samples may be due to the presence of organophosphate complexes and alterations in microbial action during protist-PGPB interactions [45].

In conclusion, the ciliate-PGPB interaction provides a piece of evidence for enhanced plant growth at the lab as well as greenhouse scale studies. The PGPB and ciliate, together, significantly enhanced the plant growth through various mechanisms. These included altering the root structure of plants, increasing the uptake of N and P by plants, causing a shift in the bacterial communities and influencing the soil respiration enzymes. This is the first report describing ciliate (Kreyellidae sp.) interaction with two different plant growth promoting rhizobacterial species, and highlighting the effect of their interaction on plant growth, soil respiration and shifts in soil microbial communities.

Ethical Approval

Not applicable

Consent to Participate

Not applicable

Consent to Publish

Not applicable

Authors Contributions

NA: designed the study and wrote the final manuscript; KAC and RSP: performed the experiment and analyzed the data; KAC: drafted the manuscript

Funding

Not applicable

Competing Interests

The authors declare that they have no conflict of interest

Availability of data and materials

All data generated or analyzed during this study are included in this published article

Acknowledgements

This work was supported by the Department of Science and Technology, Science and Engineering Research Board (DST-SERB), New Delhi, India, under ECRA grant for researchers to NA (ECR/2017/001977).

Bakker PA, Berendsen RL, Doornbos RF, Wintermans PC, Pieterse CM (2013) The rhizosphere revisited: root microbiomics. Front Plant Sci 4:165. https://doi.org/10.3389/fpls.2013.00165
Jacoby R, Peukert M, Succurro A, Koprivova A, Kopriva S (2017) The role of soil microorganisms in plant mineral nutrition—current knowledge and future directions. Front Plant Sci 8:1617. https://doi.org/10.3389/fpls.2017.01617
Basu A, Prasad P, Das SN, Kalam S, Sayyed RZ, Reddy MS, El Enshasy H (2021) Plant growth promoting rhizobacteria (PGPR) as green bioinoculants: recent developments, constraints, and prospects. Sustainability 13:1140. https://doi.org/10.3390/su13031140
Gao Z, Karlsson I, Geisen S, Kowalchuk G, Jousset A (2019) Protists: puppet masters of the rhizosphere microbiome. Trends Plant Sci 24:165–176. https://doi.org/10.1016/j.tplants.2018.10.011
Bonkowski M, Brandt F (2002) Do soil protozoa enhance plant growth by hormonal effects? Soil Biol Biochem 34:1709–1715. https://doi.org/10.1016/S0038-0717(02)00157-8
Alphei J, Bonkowski M, Scheu S (1996) Protozoa, Nematoda and Lumbricidae in the rhizosphere of Hordelymus europeaus (Poaceae): faunal interactions, response of microorganisms and effects on plant growth. Oecologia 106:111–126. https://doi.org/10.1007/BF00334413
Martínez-Reyes CM, Zaragoza SR, Cabirol N, Alarcón A, Mendoza-López MR (2021) Effect of predation by Colpoda sp. in nitrogen fixation rate of two free-living bacteria. Microb Ecol. https://doi.org/10.1007/s00248-021-01813-9
Levrat P, Pussard M, Alabouvette C (1992) Enhanced bacterial metabolism of a Pseudomonas strain in response to the addition of culture filtrate of a bacteriophagous amoeba. Eur J Protistol 28:79–84. https://doi.org/10.1016/S0932-4739(11)80322-6
Weidner S, Latz E, Agaras B, Valverde C, Jousset A (2017) Protozoa stimulate the plant beneficial activity of rhizospheric pseudomonads. Plant Soil 410:509–515. https://doi.org/10.1007/s11104-016-3094-8
Mazzola M, De Bruijn I, Cohen MF, Raaijmakers JM (2009) Protozoan-induced regulation of cyclic lipopeptide biosynthesis is an effective predation defense mechanism for Pseudomonas fluorescens. Appl Environ Microbiol 75:6804–6811. https://doi.org/10.1128/AEM.01272-09
Jousset A, Bonkowski M (2010) The model predator Acanthamoeba castellanii induces the production of 2, 4, DAPG by the biocontrol strain Pseudomonas fluorescens Q2-87. Soil Biol Biochem 42:1647–1649. https://doi.org/10.1016/j.soilbio.2010.05.018
Jousset A (2017) Application of protists to improve plant growth in sustainable agriculture. In: Mehnaz S (ed) Rhizotrophs: Plant Growth Promotion to Bioremediation. Springer, Singapore, pp 263–273. http://doi.org/10.1007/978-981-10-4862-3_13
Weller DM, Mavrodi DV, van Pelt JA, Pieterse CM, van Loon LC, Bakker PA (2012) Induced systemic resistance in Arabidopsis thaliana against Pseudomonas syringae pv. tomato by 2, 4-diacetylphloroglucinol-producing Pseudomonas fluorescens. Phytopathol 102:403–412. https://doi.org/10.1094/PHYTO-08-11-0222
Saleem M, Fetzer I, Dormann CF, Harms H, Chatzinotas A (2012) Predator richness increases the effect of prey diversity on prey yield. Nat Communicat 3:1–7. https://doi.org/10.1038/ncomms2287
Hünninghaus M, Koller R, Kramer S, Marhan S, Kandeler E, Bonkowski M (2017) Changes in bacterial community composition and soil respiration indicate rapid successions of protist grazers during mineralization of maize crop residues. Pedobiologia 62:1–8. https://doi.org/10.1016/j.pedobi.2017.03.002
Kuppardt A, Fester T, Härtig C, Chatzinotas A (2018) Rhizosphere protists change metabolite profiles in Zea mays. Front Microbiol 9:857. https://doi.org/10.3389/fmicb.2018.00857
Hahn MW, Höfle MG (2001) Grazing of protozoa and its effect on populations of aquatic bacteria. FEMS Microbiol Ecol 35:113–121. https://doi.org/10.1111/j.1574-6941.2001.tb00794
Patel P, Shah R, Joshi B, Ramar K, Natarajan A (2019) Molecular identification and biocontrol activity of sugarcane rhizosphere bacteria against red rot pathogen Colletotrichum falcatum. Biotechnol Rep 21:e00317. https://doi.org/10.1016/j.btre.2019.e00317
Patel DH, Naik JH, Amaresan N (2018) Synergistic effect of root-associated bacteria on plant growth and certain physiological parameters of banana plant (Musa acuminata). Arch Agron Soil Sci 64:1021–1031. https://doi:10.1080/03650340.2017.1410703
Lee JJ, Soldo AT (1992) Protocols in protozoology. Society of Protozoologists
Puitika T, Kasahara Y, Miyoshi N, Sato Y, Shimano S (2007) A taxon specific oligonucleotide primer set for PCR-based detection of soil ciliate. Microbes Environ 22:78–81. https://doi.org/10.1264/jsme2.22.78
Song C, Mazzola M, Cheng X, Oetjen J, Alexandrov T, Dorrestein P, Watrous J, van Der Voort M, Raaijmakers JM (2015) Molecular and chemical dialogues in bacteria-protozoa interactions. Sci Rep 5:1–13. https://doi.org/10.1038/srep12837
Kreuzer K, Adamczyk J, Iijima M, Wagner M, Scheu S, Bonkowski M (2006) Grazing of a common species of soil protozoa (Acanthamoeba castellanii) affects rhizosphere bacterial community composition and root architecture of rice (Oryza sativa L.). Soil Biol Biochem 38:1665–1672. https://doi.org/10.1016/j.soilbio.2005.11.027
Cortés-Pérez S, Rodríguez-Zaragoza S, Mendoza-López MR (2014) Trophic structure of amoeba communities near roots of Medicago sativa after contamination with fuel oil No. 6. Microb Ecol 67:430–442. http://doi.org/10.1007/s00248-013-0305-1
Gebremikael MT, Buchan D, De Neve S (2014) Quantifying the influences of free-living nematodes on soil nitrogen and microbial biomass dynamics in bare and planted microcosms. Soil Biol Biochem 70:131–141. https://doi.org/10.1016/j.soilbio.2013.12.006
Hogue E, Wilcox GE, Cantliff DJ (1970) Effect of soil phosphorous levels on phosphate fractions in tomato leaves. J Am Soc Hortic Sci 95:174–176
Frostegard A, Baath E, Tunlid A (1993) Shifts in the structure of soil microbial communities in limed forests as revealed by phospholipid fatty acid analysis. Soil Biol Biochem 25:723–730. https://doi.org/10.1016/0038-0717(93)90113-P
Adam G, Duncan H (2001) Development of a rapid and sensitive method for the measurement of total microbial activity using fluorescein diacetate (FDA) in a range of soils. Soil Biol Biochem 33:943–951. https://doi.org/10.1016/S0038-0717(00)00244-3
Casida LE, Klein DA, Santoro T (1964) Soil dehydrogenase activity. Soil Sci 98:371–376
Bessey OA, Lowery OH, Brock MJ (1946) A method of rapid determination of alkaline phosphatase with five cubic millimeters of serum. J Biol Chem 164:321–329
Anderson JM, Ingram JSI (1993) A handbook of methods. CAB International, Wallingford, Oxfordshire 221:62–65
Pedersen AL, Nybroe O, Winding A, Ekelund F, Bjørnlund L (2009) Bacterial feeders, the nematode Caenorhabditis elegans and the flagellate Cercomonas longicauda, have different effects on outcome of competition among the Pseudomonas biocontrol strains CHA0 and DSS73. Microb Ecol 57:501–509. https://doi.org/10.1007/s00248-008-9455-y
Amacker N, Gao Z, Agaras BC, Latz E, Kowalchuk GA, Valverde CF, Jousset A, Weidner S (2020) Biocontrol traits correlate with resistance to predation by protists in soil pseudomonads. Front Microbiol 11:614194. https://doi.org/10.3389/fmicb.2020.614194
Asiloglu R, Shiroishi K, Suzuki K, Turgay OC, Murase J, Harada N (2020) Protist-enhanced survival of a plant growth promoting rhizobacteria, Azospirillum sp. B510, and the growth of rice (Oryza sativa L.) plants. Appl Soil Ecol 154:103599. https://doi.org/10.1016/j.apsoil.2020.103599
Herdler S, Kreuzer K, Scheu S, Bonkowski M (2008) Interactions between arbuscular mycorrhizal fungi (Glomus intraradices, Glomeromycota) and amoebae (Acanthamoeba castellanii, Protozoa) in the rhizosphere of rice (Oryza sativa). Soil Biol Biochem 40:660–668. https://doi.org/10.1016/j.soilbio.2007.09.026
Bonkowski M (2004) Protozoa and plant growth: the microbial loop in soil revisited. New Phytol 162:617–631. https://doi.org/10.1111/j.1469-8137.2004.01066
Krome K, Rosenberg K, Dickler C, Kreuzer K, Ludwig-Müller J, Ullrich-Eberius C, Scheu S, Bonkowski M (2010) Soil bacteria and protozoa affect root branching via effects on the auxin and cytokinin balance in plants. Plant Soil 328:191–201. https://doi.org/10.1007/s11104-009-0101-3
Bonkowski M, Clarholm M (2012) Stimulation of plant growth through interactions of bacteria and protozoa: testing the auxiliary microbial loop hypothesis. Acta Protozool 51:237–247. http://doi.org/10.4467/16890027AP.12.019.0765
Gupta G, Parihar SS, Ahirwar NK, Snehi SK, Singh V (2015) Plant growth promoting rhizobacteria (PGPR): current and future prospects for development of sustainable agriculture. J Microb Biochem Technol 7:096–102. http://dx.doi.org/10.4172/1948-5948.1000188
Rosenberg K, Bertaux J, Krome K, Hartmann A, Scheu S, Bonkowski M (2009) Soil amoebae rapidly change bacterial community composition in the rhizosphere of Arabidopsis thaliana. ISME J 3:675–684. https://doi.org/10.1038/ismej.2009.11
Henkes GJ, Kandeler E, Marhan S, Scheu S, Bonkowski M (2018) Interactions of mycorrhiza and protists in the rhizosphere systemically alter microbial community composition, plant shoot-to-root ratio and within-root system nitrogen allocation. Front Environ Sci 6:117. https://doi.org/10.3389/fenvs.2018.00117
Montagnes DJ, Barbosa AB, Boenigk J, Davidson K, Jürgens K, Macek M, Parry JD, Roberts EC, Simek K (2008) Selective feeding behaviour of key free-living protists: avenues for continued study. Aquat Microb Ecol 53:83–98. https://doi.org/10.3354/ame01229
Flues S, Bass D, Bonkowski M (2017) Grazing of leaf-associated Cercomonads (Protists: Rhizaria: Cercozoa) structures bacterial community composition and function. Environ Microbiol 19:3297–3309. https://doi.org/10.1111/1462-2920.13824
Usmani Z, Kumar V, Gupta P, Gupta G, Rani R, Chandra A (2019) Enhanced soil fertility, plant growth promotion and microbial enzymatic activities of vermicomposted fly ash. Sci Rep. 9:10455. https://doi.org/10.1038/s41598-019-46821-5
Alef K, Nannipieri P, Trazar-Cepeda C (1995) Phosphatase activity. In: Alef K, Nannipieri P (eds) Methods in applied soil microbiology and biochemistry. Academic Press, London, pp 335–344

Table 1. Impact of PGPB-ciliate interaction on plant growth in the plate assay

Treatment	Shoot length (cm)	Primary root length (cm)	# Seminal roots/plant	# Lateral roots/plant
Control	9.16±0.60^b	7.60±0.32^d	3.33±0.33^d	24.0±6.24^e
Ps	8.56±0.26^b	11.86±0.44^ab	4.67±0.66^c	41.0±12.00^d
C5Ps	9.53±0.48^ab	12.53±0.31^a	9.67±0.33^b	89.3±11.67^b
Ec	6.10±0.63^c	9.43±2.23^c	5.67±0.88^c	53.0±13.45^c
C5Ec	10.30±0.80^a	12.60±1.10^a	11.67±0.88^a	115.3±22.18^a

#- numbers; Data obtained are means of three replicates and analyzed using one-way analysis of variance (ANOVA). Mean values (± S.E.) in similar column with different lower-case are significantly different (Tukey’s HSD, p<0.05). Ps-Pseudomonas sp; C5Ps- Pseudomonas sp and Kreyellidae sp; Ec- Enterobacter sp; C5Ec- Enterobacter sp and Kreyellidae sp.

Table 2. Plant growth promotion activity of PGPR-ciliate interaction in the pot experiment

Treatment	Shoot length (cm)	Root length (cm)	Shoot dry weight (g)	Root dry weight (g)
Control	19.6±1.13^d	8.3±0.47^e	0.10±0.08^e	0.05±0.06^c
Ps	30.5±1.33^c	14.9±1.48^d	0.18±0.08^d	0.09±0.08^ab
C5Ps	31.7±0.90^b	25.5±2.89^b	0.29±0.05^c	0.16±0.04^a
Ec	30.7±0.87^c	17.9±1.63^c	0.30±0.02^ab	0.08±0.06^ab
C5Ec	35.1±1.03^a	32.8±1.30^a	0.34±0.03^a	0.16±0.02^a

Data obtained are means of three replicates and analyzed using one-way analysis of variance (ANOVA). Mean values (± S.E.) in similar column with different lower-case are significantly different (Tukey’s HSD, p<0.05). Ps-Pseudomonas sp; C5Ps- Pseudomonas sp and Kreyellidae sp; Ec- Enterobacter sp; C5Ec- Enterobacter sp and Kreyellidae sp.

Table 3. Enumeration of bacterial (CFU) and protists (MPN) population in the rhizosphere soils

Treatment	CFUs g^-1	MPN g^-1
Control	15.0±0.00^d ×10⁶	1.03±0.02^d
Ps	29.4±0.01^c×10⁸	1.12±0.16^d
C5Ps	24.10±2.19^c ×10¹⁰	10.52±0.39^a
Ec	117.0±0.20^b ×10⁹	1.95±0.03^d
C5Ec	131.0±3.70^a ×10¹¹	5.12±0.06^c

Table 4. Effect of PGPR-ciliate interaction on the MBC and soil respiration activity

Treatments	Microbial carbon biomass (mg C g^-1 soil)	Alkaline phsophatase (μg p-NP g^-1 soil h^-1)	FDA analysis (μg fluorescein g^-1 soil h^-1)	Dehydrogenase (µg TPF g^-1 soil h^-1)
Control	36.88 ± 2.11^c	14.75±0.40^d	4.13±0.16^b	14.02±0.55^c
Ps	109.58 ± 4.67^b	28.39±0.37^a	5.46±0.23^a	14.60±0.31^c
C5Ps	119.34 ± 3.43^b	25.96±0.56^b	3.04±0.06^c	19.76±0.43^a
Ec	100.9 ± 2.84^b	25.06±0.73^b	4.08±0.04^b	16.78±0.52^b
C5Ec	151.42 ± 6.07^a	18.69±0.32^c	2.92±0.07^c	10.96±0.38^d

Table 5. Effect of PGPR-ciliate interaction on plant N and P uptake

Treatment	Nitrogen (%)	Phosphorous (%)
Control	1.66±0.22^b	0.173±0.012^bc
Ps	2.66±0.14^a	0.201±0.002^b
C5Ps	2.78±0.09^a	0.155±0.003^c
Ec	2.24±0.17^ab	0.108±0.002^d
C5Ec	2.76±0.11^a	0.273±0.015^a

Download PDF

Version 1

posted

You are reading this latest preprint version

Interaction of Kreyellidae sp. and plant growth promoting bacteria influences the soil characteristics and root structure of rice plants

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Microorganisms and culture conditions

Growth Assessment Assay Of Ciliate Species With Pgpb

Impact of ciliate-PGPB interaction on plant growth

Plate assay

Pot Assay

Analysis Of Soil Samples

Results

Discussion

Declarations

References

Tables

Status:

Version 1