Improvement of Verticillium Wilt Resistance by Applying Arbuscular Mycorrhizal Fungi to a Cotton Variety with High Symbiotic Efficiency under Field Conditions

Abstract

Arbuscular mycorrhizal fungi (AMF) play an important role in nutrient cycling processes and plant stress resistance. To evaluate the effect of Rhizophagus irregularis CD1 on plant growth promotion (PGP) and Verticillium wilt disease, the symbiotic efficiency of AMF (SEA) was first investigated over a range of 3% to 94% in 17 cotton varieties. The high-SEA subgroup had significant PGP effects in a greenhouse. From these results, the highest-SEA variety of Lumian 1 was selected for a two-year field assay. Consistent with the performance from the greenhouse, the AMF-mediated PGP of Lumian 1 also produced significant results, including an increased plant height, stem diameter, number of petioles, and phosphorus content. Compared with the mock treatment, AMF colonization obviously inhibited the symptom development of Verticillium dahliae and more strongly elevated the expression of pathogenesis-related genes and lignin synthesis-related genes. These results suggest that AMF colonization could lead to the mycorrhiza-induced resistance (MIR) of Lumian 1 to V. dahliae. Interestingly, our results indicated that the AMF endosymbiont could directly inhibit the growth of phytopathogenic fungi including V. dahliae by releasing undefined volatiles. In summary, our results suggest that stronger effects of AMF application result from the high-SEA.

Keywords: Gossypium hirsutum; Verticillium wilt; antifungal activity; mycorrhizal colonization; plant growth promotion; resistance; symbiotic efficiency.

Figure 1

Evaluation of the symbiotic efficiency of AMF (SEA) among seventeen cotton cultivars. One hundred 1-cm root fragments were investigated from each plant after 40 days of growth with Rhizophagus irregularis CD1 during greenhouse conditions. The parameters Hyphae only (%), Vesicles (%), and Total AMF (%) denote the frequency of internal hyphae (without vesicles), vesicles, and total mycorrhizal colonization, respectively. The experiment was repeated twice under greenhouse conditions. Error bars represent ± SD.

Figure 2

Effects of different symbiotic efficiency of AMF (SEA) on cotton growth promotion at 60 days post inoculation (dpi) under greenhouse conditions. +AMF: mycorrhizal; −AMF: nonmycorrhizal. (A) The growth phenotypes of two cotton subgroups. The low-SEA subgroup (SEA ≤ 26%) was comprised of Jiaxing 1, Lumianyan 22, Aizimian Sj-1, and Xinmian 33B. The high-SEA subgroup (SEA ≥ 66%) was comprised of Lumian 1, Binbei, Hai 3-79, and Yuzao 1; (B–D) Biomass statistics of two cotton subgroups including plant height (B), shoot fresh weight (C), and root fresh weight (D). Error bars represent ± SD. * p < 0.05; ** p < 0.01.

Figure 3

Effects of AMF on the growth of Lumian 1 at 40 dpi under field conditions. +AMF: mycorrhizal; −AMF: nonmycorrhizal. (A) The growth phenotypes of Lumian 1. (B–E) Biomass statistics of Lumian 1 including plant height (B), stem diameter (C), the number of petioles (D), and the area of the largest true leaf (E). Error bars represent ± SD. * p < 0.05; ** p < 0.01.

Figure 4

Effects of AMF on inorganic phosphorus (Pi) transport of Lumian 1 at 60 dpi under field conditions. (A) Pi content of both the root and leaf. +AMF: mycorrhizal; −AMF: nonmycorrhizal. Error bars represent ±SD. * p < 0.05; ** p < 0.01. (B) Homology tree for amino acid sequences of AtPht 1–5 (At2G32830) and its homologs within Gossypium hirsutum (Gh_A02G0202, Gh_A02G0203, Gh_D02G0263 and Gh_D10G1372), Oryza sativa (OsPT8, AAN39049; OsPT12, AAN39053), Zea mays (GRMZM2G326707, GRMZM2G154090) and Medicago truncatula (MTR_1g074930). This was generated by DNAMAN version 5.2.2.0 (Lynnon Biosoft, San Ramon, CA, USA); (C,D) Expression level of cotton homologs of AtPht 1–5 in root (C) and leaf (D). The test performed by quantitative RT-PCR analysis. Transcript abundance of genes was normalized to that of the reference gene UBQ7 (GenBank Accession Number: DQ116441). Three biological replicates were used for each reaction with three technical replicates each. Mean values and standard errors were calculated from three biological replicates.

Figure 5

Effects of AMF on the Verticillium wilt resistance of Lumian 1 under field conditions. +AMF: mycorrhizal; −AMF: nonmycorrhizal. (A) The phenotypes of Lumian 1 at the early occurrence phase of Verticillium wilt; (B) The resistance phenotypes of Lumian 1 at 120 dpi in 2016; (C) The vascular discoloration phenotypes of Lumian 1 at 150 dpi in 2016; (D,E) The disease index of leaves at 120 dpi in 2015 (D) and 2016 (E), respectively. At least 40 plants were used for each experiment; (F) Quantitative detection of the V. dahliae biomass relative to cotton leaves at 120 dpi in 2016. The average fungal biomass was determined using at least 10 mycorrhiza-treated and 10 control cottons of each plot; (G) The disease index of the vascular bundle at 150 dpi in 2016. Error bars represent ± SD. * p < 0.05; ** p < 0.01.

Figure 6

Expression patterns of cotton resistance-related genes in mycorrhizal (+AMF) and nonmycorrhizal (−AMF) Lumian 1. (A) Expression of PR genes; (B) Expression of JA synthesis-related genes; (C) Expression of lignin synthesis-related genes. The test was performed using reverse transcription quantitative PCR analysis of relative gene expression. Transcript abundance of genes was normalized to that of the reference gene UBQ7 (GenBank Accession Number: DQ116441). Three biological replicates were used for each reaction with three technical replicates each. Mean values and standard errors were calculated from three biological replicates. * p < 0.05; ** p < 0.01.

Figure 7

In vitro antimicrobial activities of AMF against Verticillium dahliae. Divided Petri dishes prevent nonvolatile solutes from diffusing between two compartments. The upper compartment of divided Petri dishes contained a complete growth medium (M+) used for the growth of AMF symbionts, and the lower compartment contained the same medium lacking sugar (M−), thus permitting the development of AMF hyphae and spores. Two discs covered with V. dahliae of 5 mm diameter were transferred to the M− compartment and incubated in the dark at 25 °C for 1 week. Before the inoculation of V. dahliae discs, fresh blank M+ and blank M− medium were decanted into divided Petri dishes. The height of M− was equal to the middle baffle while slightly higher than M+. The heights of M+ and M− in each plate were comparable to each other. Treatments were divided as follows: (A) empty M− and empty M+; (B) M− with AMF hyphae and spores (h + s) and empty M+; (C) empty M− and M+ with mycorrhizal roots (myc-roots); (D) M− with AMF h + s and M+ with myc-roots; (E) empty M− and M+ with dead myc-roots heated at 65 °C for 30 min; (F) empty M− and M+ with non-mycorrhizal roots (nm-roots). Experiments were repeated three times with similar results.

Figure 8

Antifungal activities of AMF symbionts against several soil-borne fungi at 7 dpi (A) and 50 dpi (B), respectively. The culture conditions were as described for Figure 7. The upper compartment contained M+ medium with or without AMF symbionts (myc-roots), and the downward compartment contained M− medium inoculated with two discs of Verticillium dahliae, Fusarium oxysporum, Fusarium graminearum and Rhizoctonia solani. Experiments were repeated three times with similar results.