Quorum sensing-mediated inter-specific conidial anastomosis tube fusion between Colletotrichum gloeosporioides and C. siamense

Abstract

Many plant pathogenic filamentous fungi undergo fusion of conidia through conidial anastomosis tubes (CATs), which is believed to facilitate horizontal gene transfer between species. We discovered a remarkable inter-specific CAT fusion between two important plant fungal pathogens Colletotrichum gloeosporioides and C. siamense. In an invitro assay, under no selection pressure, the inter-specific CAT fusion was preferred with higher frequency (25% ± 5%) than intra-specific CAT fusion (11% ± 3.6%). Different stages of CAT fusion viz. CAT induction, homing, and fusion were observed during this inter-specific CAT fusion. The CAT fusion was found to be higher in absence of nutrients and under physiological stresses. This CAT fusion involved a quorum sensing phenomenon, wherein the CAT induction was dependent on conidial density and the putative quorum sensing molecule was extractable in chloroform. Movement of nuclei, mitochondria, and lipid droplets were observed during the CAT fusion. Post CAT fusion, the resulting conidia gave rise to putative heterokaryotic progenies with variable colony characteristics as compared to their parental strains. Few heterokaryons showed variable AFLP banding pattern compared to their parental strains, thereby suggesting a possible genetic exchange between the two species through CAT fusion. The heterokaryotic progenies exhibited varied fitness under different stress conditions. Our study illustrated a possible role of inter-specific CAT fusion in generation of genetic and phenotypic diversity in these fungal pathogens.

Keywords: Colletotrichum; Conidial anastomosis tube; Genetic diversity; Heterokaryosis; Hybrid vigour; Quorum sensing.

Fig. 1

ApMAT gene-based phylogenetic placement of C. gloeosporioides (CBS 953.97) and C. siamense (NFCCI 3061) with the closely related species. The tree was constructed using the neighbor joining method in MEGA 7. The scale bar indicates the number of expected substitutions per site. The numbers provided on branches are frequencies with which a given branch appeared in 1000 bootstrap replications. The tree was rooted with C. xanthorrhoeae

Fig. 2

Intra and inter-specific CAT fusion in water in C. gloeosporioides and C. siamense. a Intra-specific CAT fusion in 17 days old C. gloeosporioides conidia. b Intra-specific CAT fusion in 17 days old C. siamense conidia. c Inter-specific CAT fusion between 17 days old C. gloeosporioides and C. siamense conidia. d Percentage CAT fusion in different aged conidia (4 × 10⁵ in 1 ml of water) viz. 6, 10, 13, 17 and 20 days of C. gloeosporioides and C. siamense 72 h post-incubation. e Percentage CAT fusion in 17 days old conidia (4 × 10⁵ in 1 ml of water) of C. gloeosporioides and C. siamense incubated for different incubation time viz. 24, 48, 72 and 96 h. f Percentage CAT fusion in 17 days old conidia (4 × 10⁵ in 1 ml of water) of C. gloeosporioides and C. siamense grown and incubated individually and in co-culture in water for 72 h. g Percentage CAT fusion in different conidial densities viz. 4 × 10², 4 × 10³, 4 × 10⁴, 4 × 10⁵ and 4 × 10⁶ of 17 days old conidia incubated in 1 ml of water for 72 h. Arrow indicates CAT fusion, asterisk (*) symbol indicates C. siamense conidia and plus (+) symbol indicates conidia of C. gloeosporioides. Cg: C. gloeosporioides and Cs: C. siamense. CAT fusion was quantified as the percentage of conidia involved in fusion. Average from 3 replicates (n = 3) and 150 conidial pairs were counted per replicate. Bar indicates standard deviation. Statistical significance of differences was analyzed by one-way ANOVA with Tukey’s multiple comparison post-hoc test (bars with the same letter are not significantly different; p ≤ 0.05). Scale Bar = 20 μm

Fig. 3

Different stages of inter-specific CAT fusion between C. gloeosporioides and C. siamense in-vitro in water. a CAT induction. b CAT fusion, c CAT homing and CAT fusion. d CAT network. Arrow indicates CAT fusion, arrowhead indicates CAT homing, asterisk (*) symbol indicates C. siamense conidia and plus (+) symbol indicates conidia of C. gloeosporioides. Scale Bar = 20 μm

Fig. 4

Effects of physiological stresses, nutrients, CAT inducers and CAT inhibitors on inter-specific CAT fusion frequency in C. gloeosporioides and C. siamense. Inter-specific CAT fusion percentage in water, PDB, oxidative stress (H₂O₂), osmotic stress (NaCl and sorbitol), heat, antifungal stress, glucose, KNO₃, combination of glucose and KNO₃, NaNO₃, MgCl₂, tryptophan, Insolution™ PD98059 (inhibitor of MAPKK pathway). Average from 3 replicates (n = 3) and 150 conidial pairs were counted per replicate. Bar indicates standard deviation. Statistical significance of differences was analyzed by one-way ANOVA with Tukey’s multiple comparison post-hoc test (bars with the same letter are not significantly different; p ≤ 0.05)

Fig. 5

Inter-specific CAT fusion in C. gloeosporioides and C. siamense is mediated through quorum sensing phenomena. a Percentage CAT fusion in young (6 days) and old (17 days) conidia in water, PDB, CAT medium supernatant, PDB + CAT medium supernatant and PDB + water. b Percentage CAT fusion in 17 days old conidia in water, CAT medium supernatant, chloroform treated CAT medium supernatant, proteinase K treated CAT medium supernatant, water re-extract of chloroform treated CAT medium supernatant, PDB + water, PDB+ chloroform treated CAT medium supernatant, PDB + proteinase K treated CAT medium supernatant, PDB + water re-extract of chloroform treated CAT medium supernatant. Water was originally used to generate the CAT medium supernatant. Average from 3 replicates (n = 3) and 150 conidial pairs were counted per replicate. Bar indicates standard deviation. Statistical significance of differences was analyzed by one-way ANOVA with Tukey’s multiple comparison post-hoc test (bars with the same letter are not significantly different; p ≤ 0.05). Scale Bar = 20 μm

Fig. 6

Transfer of nucleus and different cell organelles during CAT fusion between C. gloeosporioides and C. siamense. a, b and c Bright-field microscopy of CAT fusion between the 17 days old conidia C. gloeosporioides and C. siamense. d, e and f Visualization of nuclear transfer between the 17 days old conidia of C. gloeosporioides and C. siamense by DAPI staining using fluorescence microscope. g Fluorescent, h Brightfield and i Overlapping microscopic images of mitochondrial movement by MitoRed staining. j Fluorescent, k Brightfield and l Overlapping microscopic images of movement of lipid droplets by Nile red staining. Arrowhead indicates nucleus, arrow indicates CAT fusion, asterisk (*) symbol indicates C. siamense conidia and plus (+) symbol indicates conidia of C. gloeosporioides. Scale bar = 20 μm for images (a-f) and 10 μm for images (g-l)

Fig. 7

Colony morphology of C. gloeosporioides and C. siamense parent strains and their post-CAT fusion progenies. Colony morphology of parent C. gloeosporioides and C. siamense strains and their putative heterokaryotic progenies CG1-CG10 and CS1-CS10, respectively derived by single spore isolation from a mixture of conidia that had undergone CAT fusion

Fig. 8

AFLP analysis of C. gloeosporioides and C. siamense parent strains and their putative heterokaryotic progenies. a AFLP banding patterns of parent C. gloeosporioides (CGP), C. siamense (CSP) and putative heterokaryotic progenies of C. gloeosporioides (CG1-CG10). b AFLP banding patterns of parent C. siamense (CSP), C. gloeosporioides (CGP) and putative heterokaryotic progenies of C. siamense (CS1-CS10) along with 100 bp DNA marker (M). Asterisk indicates the presence of extra bands beneath them

Fig. 9

Growth rate (cm) of C. gloeosporioides parent (CGP) and their heterokaryotic progenies (CG1–10) under different stresses. a Growth rate in presence of oxidative stress induced by H₂O₂. b Growth rate in presence of osmotic stress induced by NaCl. c Growth rate in osmotic stress induced by sorbitol. Average from 3 replicates and bar indicates standard deviation. Statistical significance of differences was analyzed by one-way Tukey’s multiple comparison post-hoc test (bars with the same letter are not significantly different; p ≤ 0.05)