The Characterization and the Biological Activity of Phytotoxin Produced by Paraphoma radicina

Abstract

Paraphoma radicina is a new pathogen that causes alfalfa paraphoma root rot (APRR), leading to alfalfa production losses. The resistance levels of 30 alfalfa cultivars to APRR have already been characterized. However, the pathogenic mechanism of P. radicina is still unclear. This study aimed to assess the effects of a crude toxin extracted from P. radicina cell-free culture filtrate (CFCF) on susceptible and resistant cultivars of alfalfa. Meanwhile, the crude toxin components were detected using gas chromatography-mass spectrometry (GC-MS) analysis. CFCF cultured in MEB medium for 14 days and crude toxin extracted by ethyl acetate induced significant phytotoxicity caused the average lesion areas of 5.8 and 3.9 mm², respectively, on alfalfa leaves. The crude toxin exhibited resistance to high temperature, as shown by a lesion area of 3.6 mm² when treated at 120 °C for 30 min. Different concentrations of the crude toxin in water and MS medium had different effects on susceptible and resistant cultivars. Moreover, the crude toxin affected the plasma membrane, mitochondria, and nuclear membranes of alfalfa root cortical cells. Further, it induced significant phytotoxicity on Sonchus oleraceus L., Capsella bursa-pastoris (Linn.) Medic, and Chenopodium album L. Agropyron cristatum L. (average lesion areas; 11.6, 15.8, 21.4, and 6.2 mm², respectively), indicating that the crude toxin of P. radicina is a non-host-selective toxin. GC-MS analysis detected four possible active substances in the toxin (3-hydroxypyridine, 5-methylresorcinol, 3-Hydroxypropionic acid, and 4-Hydroxyphenylethanol). Therefore, this study may provide insight into the pathogenic mechanism of P. radicina to alfalfa.

Keywords: Paraphoma radicina; alfalfa root rot; pathogenic activities; phytotoxin.

Figure 1

Phytotoxicity for cell-free culture filtrate (CFCF) produced by Paraphoma radicina that cultured in different medium and times. The mean lesion area was calculated from six leaves of tissue-cultured alfalfa plantlets (n = 6). Values (means + SE) marked with different letters in each column indicate significant differences (p < 0.05) based on one-way ANOVA followed by Duncan’s multiple range test. P_M-values, P_D-values, and P_M*D-values of the ANOVA indicate significant differences p < 0.05 (independent t-test) culture medium (M), incubation days (D), and their interaction (M*D), respectively.

Figure 2

The crude toxin phytotoxicity of solvent extraction by different organic. The mean lesion area was calculated from six leaves of tissue-cultured alfalfa plantlets (n = 6). Values (means + SE) marked with different letters in each column indicate significant differences (p < 0.05) based on one-way ANOVA followed by Duncan’s multiple range test.

Figure 3

The phytotoxicity of crude toxin after treated by different temperature. The mean lesion area was calculated from six leaves of tissue-cultured alfalfa plantlets (n = 6). Values (means + SE) marked with different letters in each column indicate significant differences (p < 0.05) based on one-way ANOVA followed by Duncan’s multiple range test.

Figure 4

The germination rate of susceptible and resistant cultivars seeds treated with different concentrations of crude toxin in water medium. (A) crude toxin effect on germination rate of susceptible cultivar. (B) crude toxin effect on germination rate of resistant cultivar. (C) the different inhibition rates of germination between the susceptible and the resistant cultivars. The test of germination rate was calculated from 90 (n = 90). Values (means + SE) marked with different letters in each column indicate significant differences (p < 0.05) based on one-way ANOVA followed by Duncan’s multiple range test. The significant different inhibition rate between susceptible and resistant cultivars, * indicate p < 0.05, ** indicate p < 0.01.

Figure 5

The radicle length of susceptible and resistant cultivars treated with different concentration of crude toxin in water medium. (A) crude toxin effect on radicle length of susceptible cultivar. (B) crude toxin effect on radicle length of resistant cultivar. (C) difference in inhibition rates of radicle length between the susceptible and resistant cultivars. The test of radicle length was calculated from 45 (n = 45). Values (means + SE) marked with different letters in each column indicate significant differences (p < 0.05) based on one-way ANOVA followed by Duncan’s multiple range test. The significant different inhibition rate between susceptible and resistant cultivars, * indicate p < 0.05, *** indicate p < 0.001.

Figure 6

The germination rate of susceptible and resistant cultivars seeds treated with different concentrations of crude toxin in MS medium. (A) crude toxin effect on germination rate of susceptible cultivar. (B) crude toxin effect on germination rate of resistant cultivar. (C) difference in inhibition rate of germination between the susceptible and the resistant cultivars. The test of germination rate was calculated from 60 (n = 60). Values (means + SE) marked with different letters in each column indicate significant differences (p < 0.05) based on one-way ANOVA followed by Duncan’s multiple range test. The significant different inhibition rate between susceptible and resistant cultivars, ** indicate p < 0.01, *** indicate p < 0.001.

Figure 7

The shoot and radicle length of susceptible and resistant cultivars treated with different concentrations of crude toxin in MS medium. (A) crude toxin effect on shoot length of susceptible cultivar. (B) crude toxin effect on shoot length of resistant cultivar. (C) crude toxin effect on radicle length of susceptible cultivar. (D) crude toxin effect on radicle length of resistant cultivar. (E) the difference in inhibition rates of shoot length between the susceptible and the resistant cultivars. (F) the difference in inhibition rates of radicle length between the susceptible and the resistant cultivars. Photograph of the experimental set up clearly exhibiting the inhibitory effect of different concentrations of crude toxin on the pre-germinated alfalfa seedling growth of susceptible (G) and resistant (H) cultivars. The test of shoot and radicle length was calculated from 45 (n = 45). Values (means + SE) marked with different letters in each column indicate significant differences (p < 0.05) based on one-way ANOVA followed by Duncan’s multiple range test. The significant different inhibition rate between susceptible and resistant cultivars, ** indicate p < 0.01, *** indicate p < 0.001.

Figure 8

Ultrastructure of root cortical cells of alfalfa susceptible and resistant cultivars treated with crude toxin (2 × 10⁻³ g/mL). (A,B) ultrastructure of root cortical cells of susceptible cultivar treated with 20% ethyl acetate for 24 h (N, nuclear. M, mitochondria). (C,D) plasma-membrane invaginations (PI) and irregular shape small vacuoles (V) in susceptible cultivar root treated with crude toxin for 3 h. (E) deformation of mitochondria with loss of cristae in susceptible cultivar root treated with crude toxin for 6 h. (F) the nuclear membranes were disrupted in susceptible cultivar root treated with crude toxin for 12 h. (G) severe plasmolysis of cells in susceptible cultivar root treated with toxin for 12 h. (H) ultrastructure of root cortical cells of resistant cultivar treated with 20% ethyl acetate for 24 h. (I) observed plasma membrane in a few cells in resistant cultivar root treated with crude toxin for 6 h. (J) deformation of mitochondria with loss of cristae in susceptible cultivar root treated with crude toxin for 12 h. (K) nuclear membrane in resistant cultivar root treated with crude toxin for 12 h. (L) the plasma membrane in resistant cultivar was exacerbated after treatment with crude toxin for 24 h.

Figure 9

Phytotoxin of crude toxin on the leaves of Sonchus oleraceus (E), Capsellabursa-pastoris (F), Chenopodium album (G), and Agropyron cristatum (H) changed after treated for 72 h. (A) control of Sonchus oleraceus. (B) control of Capsellabursa-pastoris. (C) control of Chenopodium album. (D) control of Chenopodium album.

Figure 10

The lesion area caused by crude toxin on Sonchus oleraceus, Capsellabursa-pastoris, Chenopodium album, and Agropyron cristatum leaves. Phytotoxic test was carried out with a concentration of 2 × 10⁻³ g/mL. The mean lesion area was calculated from three leaves (n = 3). Values (means + SE) marked with different letters in each column indicate significant differences (p < 0.05) based on one-way ANOVA followed by Duncan’s multiple range test.