Screening of Alfalfa Varieties Resistant to Phytophthora cactorum and Related Resistance Mechanism

Abstract

Alfalfa is one of the most important legume forages in the world. Root rot caused by soil-borne pathogens severely restricts the production of alfalfa. The knowledge of the interaction between alfalfa and root rot-pathogens is still lacking in China. Phytophthora cactorum was isolated from symptomatic seedlings of an alfalfa field in Nanjing with high levels of damping-off. We observed the different infection stages of P. cactorum on alfalfa, and found that the purified P. cactorum strain was aggressive in causing alfalfa seed and root rot. The infecting hyphae penetrated the epidermal cells and wrapped around the alfalfa roots within 48 h. By evaluating the resistance of 37 alfalfa cultivars from different countries to P. cactorum, we found Weston is a resistant variety, while Longdong is a susceptible variety. We further compared the activities of various enzymes in the plant antioxidant enzyme system between Weston and Longdong during P. cactorum infection, as well as gene expression associated with plant hormone biosynthesis and response pathways. The results showed that the disease-resistant variety Weston has stronger antioxidant enzyme activity and high levels of SA-responsive PR genes, when compared to the susceptible variety Longdong. These findings highlighted the process of interaction between P. cactorum and alfalfa, as well as the mechanism of alfalfa resistance to P. cactorum, which provides an important foundation for breeding resistant alfalfa varieties, as well as managing Phytophthora-caused alfalfa root rot.

Keywords: Phytophthora cactorum; alfalfa; resistance; root rot.

Figure 1

Morphological characteristics of Phytophthora cactorum. (A) Morphology of hyphae. (B) Pyriform sporangia. (C) Sporangia that are releasing zoospores. (D) Zoospores. (E) Empty sporangium. (F) Oospore. Arrows indicate morphological characteristics. Scale bars: 40 μm.

Figure 2

Molecular phylogenetic analysis by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The tree with the highest log likelihood (−3414.8106) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 15 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position.

Figure 3

Symptoms, survival rates, and fresh weight of alfalfa inoculated with or without Phytophthora cactorum. (A) Growth phenotypes of alfalfa inoculated with P. cactorum or not in pot. (B) Survival rates and (C) fresh weight of alfalfa inoculated with P. cactorum or not. Data are the means of six independent experiments and error bars represent SD. Asterisks at the top of the bars indicate statistical significance (T-test, p < 0.05).

Figure 4

Photo of alfalfa root staining after Phytophthora cactorum infection. (A,B) Invasive hypha grows intercellularly in epidermal cells. Different root tissues were stained with the lactophenol-trypan blue for 30 s before microscope observation. Infectious growth was observed at 24 hpi. (C) Invasive hypha grows in/outside of alfalfa cells. Infectious growth was observed at 36 hpi. (D) Typical infection sites of alfalfa roots inoculated with P. cactorum strain, showing stained mycelium wrapped around alfalfa roots and proliferation within tissue. Infectious growth was observed at 48 hpi. Scale bars = 100 μm. The arrow indicates typical infection hypha.

Figure 5

Performance of different alfalfa varieties after Phytophthora cactorum infection. Performance index (Fresh weights of 2-week-old plants normalized by germination rate) of alfalfa plants inoculated with P. cactorum. At least three independent biological replicates were performed for each treatment. Plant performance index (PPI) (PPI = Relative fresh weights (RFW) × Relative survival rate (RSR).

Figure 6

Microscopic observation of resistant or susceptible alfalfa varieties infected by Phytophthora cactorum at different time points. (A–D) Typical infection sites of Longdong roots inoculated with the P. cactorum strain, showing greater mycelium proliferation and tissue invasion within tissue. (E–H) The infection sites of Weston infected with P. cactorum, showing less mycelial infection. Different root tissues were stained with the lactophenol-trypan blue for 30 s before microscope observation. Infectious growth was observed from 6 hpi to 24 hpi. Scale bars = 200 μm.

Figure 7

Antioxidant enzyme activities of resistant or susceptible alfalfa varieties infected by Phytophthora cactorum at different time points. (A) Peroxidase (POD), (B) Superoxide dismutase (SOD), (C) Phenylalanine ammonia-lyase (PAL), (D) Polyphenol oxidase (Ppo) were measured at 0, 12, 24 or 48 hpi. Data are the means of six independent experiments and error bars represent SD. Asterisks at the top of the bars indicate statistical significance (T-test, p < 0.05).

Figure 8

Relative expression of plant hormone-related genes in resistant or susceptible alfalfa varieties infected by Phytophthora cactorum at different time points. Expression of (A) MsPR1, (B) MsPR2, (C) MsERF1, (D) MsLOX1 were detected at 0, 12, 24, or 48 hpi. Data are the means of six independent experiments and error bars represent SD. Asterisks at the top of the bars indicate statistical significance (T-test, p < 0.05).