Development of an inoculant of phosphate rock-solubilizing bacteria to improve maize growth and ...

148 Pages·2014·2.19 MB·English

by  Magallón Servin, Paola

DEVELOPMENT OF AN INOCULANT OF PHOSPHATE
ROCK-SOLUBILIZING BACTERIA TO IMPROVE MAIZE
GROWTH AND NUTRITION

Thèse
Paola Magallón Servín
Doctorat en microbiologie agroalimentaire -
Philosophiae doctor (Ph.D.)

Québec, Canada

© Paola Magallón Servín, 2014

Résumé

L'utilisation directe de roche phosphatée (RP) est une alternative viable pour remplacer les coûteux fertilisants
chimiques dans les pays en voie de développement. L'utilisation de bactéries solubilisatrices de RP (BSRP)
est un bon moyen pour augmenter la réactivité de la RP. L'objectif principal de ce travail a été d'obtenir des
isolats provenant de la mycorhizosphère du maïs possédant une grande capacité de solubilisation de RP,
compatibles avec la mycorhize arbusculaire (MA) et présentant des traits associés aux rhizobactéries
favorisant la croissance de plantes (RFCP) pour le développement d'un inoculant bactérien pour le maïs.À
partir de 118 isolats obtenus de maïs biologique cultivé au Québec, huit BSRP ont été identifiés comme Asaia
lannaensis Vb1, Pseudomonas sp. Vr14, Rahnella aquatilis (Vr7, Vr13 et Sr24) et Pantoea agglomerans (Vr9,
Ve16 et Vr25). En milieu liquide, les isolats ont dissous le P des RP selon leur réactivité
(Gafsa>Tilemsi>Maroc). La solubilisation des RP s'est effectuée par la production d'acides organiques (OA) et
l'abaissement du pH. Les meilleures BSRP de chaque groupe: (A. lannaensis Vb1, Pseudomonas sp. Vr14, R.
aquatilis Sr24 et P. agglomerans Vr25) ont été sélectionnées selon leur capacité élevée de solubilisation de la
RP et de leur production d'acide indolacétique (AIA) et de sidérophores. L‟importance des biofilms formés,
ainsi que le degré de motilité variaient selon les isolats et tous étaient compatibles avec le Glomus irregulare
(Gi). L‟étude de la colonisation des racines montre que R. aquatilis Sr24 et P. agglomerans Vr25 ont été les
meilleurs colonisateurs. Lors des expériences en serre, certains mélanges contenant R. aquatilis Sr24, P.
agglomerans Vr25 et Gi, ont amélioré la biomasse, l'absorption des nutriments et la colonisation de la plante
en association avec un champignon mycorhizien indigène du maïs cultivé dans un sol non stérile et fertilisé
avec la RP Marocaine. Nous attribuons ces résultats à leur capacité d'être de bonnes BSRP colonisatrices des
racines. Elles sont aussi compatibles avec Gi et sont capables de produire de l'AIA et des sidérophores. Cette
thèse démontre donc le potentiel d'utilisation de BSRP comme inoculant afin d'améliorer l'efficacité de
l'utilisation directe de RP comme fertilisant phosphaté pour l'agriculture durable du maïs.

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Abstract

Phosphorous is the second most important nutrient for plant growth, but its availability is often reduced.
Therefore high quantities of expensive soluble P-fertilizers are added to soil. Direct use of phosphate rock (PR)
is an alternative to chemical P-fertilizers in developing countries and for sustainable agriculture. In order to
increase PR reactivity the use of phosphate rock-solubilizing bacteria (PRSB) is a good alternative. Therefore,
the main objective of this work was to obtain mycorrhizosphere-competent PRSB presenting other PGPR-
associated traits to be used for the development of an inoculant to improve maize growth and P nutrition. Out
of 118 isolates obtained from organically grown maize in Quebec, eight PRSB were identified as Asaia
lannaensis Vb1, Pseudomonas sp. Vr14, Rahnella aquatilis (Vr7, Vr13 and Sr24) and Pantoea agglomerans
(Vr9, Ve16 and Vr25). All isolates were able to mobilize P from different sparingly soluble P sources in solid
media. In liquid medium the isolates were able to solubilize P from PRs according to their reactivity
(Gafsa>Tilemsi>Morocco). PRs were solubilized by the production of organic acids (OAs) and by lowering the
pH. The best PRSB from each group (A. lannaensis Vb1, Pseudomonas sp. Vr14, R. aquatilis Sr24 and P.3
agglomerans Vr25) were selected based on their high PR solubilization, and capacity for indolacetic acid (IAA)
and siderophore production. These four isolates presented different biofilm formation and motility capacities
and were compatible with Glomus irregulare (Gi). A root colonization study showed that R. aquatilis Sr24 and
P. agglomerans Vr25 were the best root colonizers. Vr25 was very competitive when used with other PRSB. In
greenhouse trials, plant inoculation with R. aquatilis Sr24 and P. agglomerans Vr25 in addition to Gi, increased
the biomass, nutrient uptake in non-sterile soil amended with Moroccan PR (MPR). We attribute these results
not only to their PR-solubilizing capacity but also for their ability to be good PRSB, competitive root colonizers,
compatible with Gi and to produce IAA and siderophores. This thesis shows that PRSB with AM fungi can be
used as inoculants to improve the efficiency of the direct use of PR as P fertilizer for sustainable maize
production.

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Table of contents
Résumé ............................................................................................................................................................... iii
Abstract ............................................................................................................................................................... v
Table of contents ................................................................................................................................................ vii
List of tables ....................................................................................................................................................... ix
List of figures ...................................................................................................................................................... xi
List of abbreviations .......................................................................................................................................... xiii
Acknowledgments ............................................................................................................................................ xvii
Foreword ........................................................................................................................................................... xix
CHAPTER I. INTRODUCTION ............................................................................................................................ 1
1.1 Inorganic phosphorus fixation and its management in agricultural soils .......................................... 1
1.2 Adsorption and fixation of organic phosphorus in soils .................................................................... 2
1.3 The use of P fertilizers and the importance of phosphate rock (PR) ................................................ 2
1.4 Phosphate solubilization by soil microorganisms ............................................................................. 3
1.5 Maize as a model to study PSM ....................................................................................................... 6
1.6 The importance of combining different PSMs to be used as biofertilizers ........................................ 6
1.7 The importance of biofilm formation in PSM .................................................................................... 9
1.8 Hypotheses .................................................................................................................................... 11
1.9 Objectives ...................................................................................................................................... 12
1.9.1 General aim ............................................................................................................................... 12
1.9.2 Specific objectives ..................................................................................................................... 12
1.10 References ..................................................................................................................................... 12
CHAPTER II. PHOSPHATE ROCK-SOLUBILIZING RHIZOBACTERIA ISOLATED FROM MAIZE (Zea mays)
MYCORRHIZOSPHERE ................................................................................................................................... 21
2.1 Introduction .................................................................................................................................... 23
2.2 Materials and methods ................................................................................................................... 25
2.2.1 Sampling and isolation of PRSB ................................................................................................ 25
2.2.2 PRSB inocula ............................................................................................................................ 26
2.2.3 Soil and PR chemical analyses ................................................................................................. 26
2.2.4 PRSB identification .................................................................................................................... 26
2.2.5 Solubilization of PRs and organic acids production by PRSB ................................................... 27
2.2.6 Effect of EDTA on bacterial solubilization of Morocco PR ......................................................... 28
2.2.7 Phosphatase and phytase production ....................................................................................... 28
2.2.8 Plant growth-promoting characteristics of the PRSB and their effect on maize seedlings ........ 29
2.2.9 Statistical analysis ..................................................................................................................... 31
2.3 Results ........................................................................................................................................... 32
2.3.1 Isolation and identification of PRSB .......................................................................................... 32
2.3.2 Solubilization of phosphate rocks by bacteria............................................................................ 33
2.3.3 Effect of EDTA-Na on Morocco PR solubilization .................................................................... 40
2
2.3.4 PRSB phosphatase and phytase activities ................................................................................ 42
2.3.5 Some in vitro characteristics of PRSB and their effects on maize seedling growth ................... 44
2.4 Discussion ...................................................................................................................................... 46
2.4.1 Isolation, and identification of PRSB, and evaluation of the selected strains for their capacity to
dissolve different inorganic P sources ..................................................................................................... 46
2.4.2 Determination of the solubilization of PR with different reactivity by PRSB ............................... 47
2.4.3 Effect of EDTA on Morocco PR solubilization ........................................................................... 52
2.4.4 PRSB ability to mineralize organic P, some in vitro PGPR characteristics of the selected
isolates and their effect on maize seedlings growth. ............................................................................... 52
2.5 Conclusion ..................................................................................................................................... 55
2.6 References ..................................................................................................................................... 55
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CHAPTER III. COMPATIBILITY OF PR-SOLUBILIZING BACTERIA WITH THE ARBUSCULAR
MYCORRHIZAL FUNGUS (Glomus irregulare) ................................................................................................ 65
3.1 Introduction ..................................................................................................................................... 67
3.2 Materials and methods ................................................................................................................... 68
3.2.1 Compatibility of the selected PRSB with AMF in two-compartment Petri dish ........................... 68
3.2.2 Compatibility of the selected PRSB with AMF under greenhouse conditions ............................ 69
3.2.3 Statistical analysis ..................................................................................................................... 69
3.3 Results ........................................................................................................................................... 70
3.3.1 Compatibility of the selected PRSB with Glomus irregulare in two-compartment Petri dish ...... 70
3.3.2 Effect of the four selected PRSB on root mycorrhization of leek by Glomus irregulare under
greenhouse conditions ............................................................................................................................. 71
3.4 Discussion ...................................................................................................................................... 72
3.5 Conclusions .................................................................................................................................... 76
3.6 References ..................................................................................................................................... 76
CHAPTER IV. DEVELOPMENT OF A COMBINED INOCULUM CONTAINING DIFFERENT PR-
SOLUBILIZING BACTERIA BENEFICIAL TO MAIZE ....................................................................................... 81
4.1 Introduction ..................................................................................................................................... 83
4.2 Material and methods ..................................................................................................................... 84
4.2.1 Bacteria ..................................................................................................................................... 84
4.2.2 Biocompatibility assay ............................................................................................................... 84
4.2.3 Determination of the PR solubilization capacity of the four different PRSB individually and in
combination .............................................................................................................................................. 84
4.2.4 Determination of specific biofilm formation capacity on abiotic surfaces by the traditional crystal
violet method ............................................................................................................................................ 85
4.2.5 Determination of biofilm formation capacity on MPR surface .................................................... 85
4.2.6 Effect of different PRSB isolates, individually and in mixture, on maize (Zea mays L. cv.
SENECA Horizon) root colonization in growth pouches........................................................................... 86
4.2.7 Greenhouse experiment to determine the effects of different mixtures of the PRSB on the
growth of forage maize (Zea mays L. cv. Focus) in the presence of the AMF Glomus irregulare ............ 87
4.2.8 Statistical analysis ..................................................................................................................... 89
4.3 Results ........................................................................................................................................... 89
4.3.1 Biocompatibility .......................................................................................................................... 89
4.3.2 Phosphate rock solubilization in liquid media by PRSB used individually or in mixtures ........... 89
4.3.3 Specific biofilm formation (SBF) index of the selected PRSB on abiotic surfaces, individually
and in mixture, calculated by the traditional crystal violet (CV) method ................................................... 94
4.3.4 Determination of biofilm formation capacity on MPR surface .................................................... 94
4.3.5 Effect of the PRSB on growth and root colonization of two-week-old maize seedlings (Zea mays
L. cv. SENECA Horizon) in growth pouches ............................................................................................ 98
4.3.6 Greenhouse experiment .......................................................................................................... 100
4.4 Discussion .................................................................................................................................... 107
4.4.1 Solubilization of PR by the selected PRSB as single or multiple species inoculants in liquid
media 107
4.4.2 Specific biofilm formation (SBF) index of the selected PRSB .................................................. 109
4.4.3 Effect of PRSB on growth and root colonization of two week old maize seedlings (Zea mays L.
cv. SENECA Horizon) in growth pouches .............................................................................................. 110
4.4.4 Greenhouse experiment .......................................................................................................... 111
4.5 Conclusion .................................................................................................................................... 113
4.6 References ................................................................................................................................... 114
GENERAL CONCLUSION .............................................................................................................................. 119
ANNEX I. Complementary material and statistical analysis ............................................................................ 121
ANNEX II. Media composition ......................................................................................................................... 127
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List of tables
Table 1. Phosphate-solubilizing fungi. ................................................................................................................ 4
Table 2. Phosphate-solubilizing bacteria. ........................................................................................................... 5
Table 3. Synergism between rhizobacteria and AM fungi in various crops. ........................................................ 8
Table 4. Phenotypic characteristics of colonies of phosphate rock-solubilizing bacteria (PRSB) and their P
dissolution activity on solid NBRIP medium supplemented with different P sources. .............................. 32
Table 5. Chemical composition of phosphate rocks and extractability of P and other elements with 2% (wt/vol)
citric or 2% (wt/vol) formic acid. ............................................................................................................... 34
Table 6. ANOVA (SAS Mixed Procedure) of the effects of Morocco, Gafsa, and Tilemsi phosphate rocks on P
solubilization by the selected PRSB in NBRIP liquid medium.................................................................. 35
Table 7. Effect of inoculation with phosphate rock-solubilizing bacteria on the pH of the culture filtrate of
NBRIP supplement with different phosphate rocks, after 5 days of growth at 28°C ................................ 37
Table 8. ANOVA (SAS MIXED procedure) of the effect of the addition of 12 mM of EDTA-Na on the Morocco
2
PR solubilization by PRSB in NBRIP liquid medium. ............................................................................... 40
Table 9. Phosphatases and phytase production by phosphate rock-solubilizing bacteria (PRSB) .................. 43
Table 10. Indolacetic acid (IAA) and siderophore production, specific biofilm formation (SBF) and swimming
motility in phosphate rock-solubilizing bacteria. ....................................................................................... 45
Table 11. Effect of inoculation with PRSB on dry matter yields of two-week old maize seedlings (Zea mays cv.
SENECA, Horizon). ................................................................................................................................. 45
Table 12. Effect of inoculation with PRSB on fresh and dry matter yields of 70-day-old leek plants (Allium
ampeloprasum var. porrum cultivar „Lancelot‟) in presence or absence of G. irregulare, under
greenhouse conditions. ............................................................................................................................ 72
Table 13. Effect of inoculation of PRSB on the percentage of Glomus irregulare root colonization of 30-, 43-
and 70-day-old leek plants under greenhouse conditions. ...................................................................... 72
Table 14. Effect of inoculation with phosphate rock-solubilizing bacteria on the pH of the culture filtrate of
NBRIP-MPR, after 7 days of growth at 28 ºC .......................................................................................... 92
Table 15. Effect of biofilm conformed by PRSB over MPR particles on the pH of the culture, after 5 days at
28ºC. Tested strains were Asaia lannaensis (Vb1), Pseudomonas sp. (Vr14), Rahnella aquatilis (Sr24)
and Pantoea agglomerans (Vr25). ........................................................................................................... 96
Table 16. Effect of inoculation with PRSB (used individually or in mixed culture) on dry matter yields of 2-
week-old maize seedlings (Zea mays cv. SENECA, Horizon). ................................................................ 99
Table 17. Summary of analyses of variance of shoot fresh and dry matter, AM colonization (% AM), and total
N, P and K uptake by corn (Zea mays cv. FOCUS) fertilized with MPR and inoculated with different
PRSB, individually and in mixture, in the presence or absence of Glomus irregulare (Gi). ................... 101
Table 18. Effect of inoculating maize (Zea mays cv. FOCUS) plants with PRSB, either individually or in
different combinations, on shoot fresh and dry matter, nutrient uptake and PRSB root colonization in a
soil amended with MPR in the presence and absence of Glomus irregulare (Gi) under greenhouse
conditions .............................................................................................................................................. 105
Table 19. Summary of analyses of variance for numbers of potential P solubilizing bacteria (PPSB) present in
the rhizosphere of maize (Zea mays cv. FOCUS) fertilized with MPR and inoculated with different PRSB,
individually and in combination, in the presence or absence of Glomus irregulare (Gi). ....................... 107

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