Population genomic analysis reveals geographic structure and climatic diversification for Macrophomina phaseolina isolated from soybean and dry bean across the United States, Puerto Rico, and Colombia

Macrophomina phaseolina causes charcoal rot, which can significantly reduce yield and seed quality of soybean and dry bean resulting from primarily environmental stressors. Although charcoal rot has been recognized as a warm climate-driven disease of increasing concern under global climate change, knowledge regarding population genetics and climatic variables contributing to the genetic diversity of M. phaseolina is limited. This study conducted genome sequencing for 95 M. phaseolina isolates from soybean and dry bean across the continental United States, Puerto Rico, and Colombia. Inference on the population structure using 76,981 single nucleotide polymorphisms (SNPs) revealed that the isolates exhibited a discrete genetic clustering at the continental level and a continuous genetic differentiation regionally. A majority of isolates from the United States (96%) grouped in a clade with a predominantly clonal genetic structure, while 88% of Puerto Rican and Colombian isolates from dry bean were assigned to a separate clade with higher genetic diversity. A redundancy analysis (RDA) was used to estimate the contributions of climate and spatial structure to genomic variation (11,421 unlinked SNPs). Climate significantly contributed to genomic variation at a continental level with temperature seasonality explaining the most variation while precipitation of warmest quarter explaining the most when spatial structure was accounted for. The loci significantly associated with multivariate climate were found closely to the genes related to fungal stress responses, including transmembrane transport, glycoside hydrolase activity and a heat-shock protein, which may mediate climatic adaptation for M. phaseolina. On the contrary, limited genome-wide differentiation among populations by hosts was observed. These findings highlight the importance of population genetics and identify candidate genes of M. phaseolina that can be used to elucidate the molecular mechanisms that underly climatic adaptation to the changing climate

Anatomical response and infection of soybean during latent and pathogenic infection by type A and B of Phialophora gregata.

Growth and anatomical responses of plants during latent and pathogenic infection by fungal pathogens are not well understood. The interactions between soybean (Glycine max) and two types of the pathogen Phialophora gregata were investigated to determine how plants respond during latent and pathogenic infection. Stems of soybean cultivars with different or no genes for resistance to infection by P. gregata were inoculated with wildtype or GFP and RFP-labeled strains of types A or B of P. gregata. Plants were sectioned during latent and pathogenic infection, examined with transmitted light or fluorescent microscopy, and quantitative differences in vessels and qualitative differences in infection were assessed using captured images. During latent infection, the number of vessels was similar in resistant and susceptible plants infected with type A or B compared to the control, and fungal infection was rarely observed in vessels. During pathogenic infection, the resistant cultivars had 20 to 25% more vessels than the uninfected plants, and fungal hyphae were readily observed in the vessels. Furthermore, during the pathogenic phase in a resistant cultivar, P. gregata type A-GFP was limited to outside of the primary xylem, while P. gregata type B-RFP was observed in the primary xylem. The opposite occurred with the susceptible cultivar, where PgA-GFP was observed in the primary xylem and PgB-RFP was limited to the interfascicular region. In summary, soybean cultivars with resistance to BSR produced more vessels and can restrict or exclude P. gregata from the vascular system compared to susceptible cultivars. Structural resistance mechanisms potentially compensate for loss of vessel function and disrupted water movement

Stem anatomy of soybean during latent and pathogenic infection by <i>Phialophora gregata</i>.

<p>Stem anatomy of the resistant soybean cultivar (Bell) and the susceptible cultivar (Corsoy 79) during latent and pathogenic infection by types A and B of <i>Phialophora gregata</i>. During latent infection (2 weeks post inoculation) no differences were seen in anatomical organization in Corsoy 79 inoculated with type A (A), type B (B) or the control (C), but differences in the vascular cambium and xylem structure was seen during pathogenic infection (6 weeks post inoculation) (D–F). During latent infection of Bell, the vascular organization was similar in plants inoculated with type A (G) and the control (I), but plants infected with type B (H) had less secondary growth and a less pronounced layer of vascular cambium. Differences in anatomical organization were observed during pathogenic infection Bell inoculated with type B (K) had less secondary growth and less of a cambium layer compared to the Bell inoculated with type A (J) and the controls (F, L). All images were taken at 40x magnification. Treatments for experiment 1 were replicated three times (n = 3 plants/replication). Treatments for experiment 3 were replicated three times (n = 4 plants/replication).</p

Average quantity of vessels of stems during latent and pathogenic infection with <i>Phialophora gregata</i>.

<p>Average quantity of vessels per cross section of stems two (A) and six (B) weeks post inoculation with either type A (PgA) or B (PgB) of <i>Phialophora gregata</i> for six soybean cultivars. Letters in parentheses next to cultivars indicate the source of BSR resistance or if the cultivar is susceptible (S). Treatments were compared using an ANOVA followed by a Fisher's LSD test, and bars with different letters are significantly different (<i>P</i> = 0.05). Error bars indicte 95% confidence intervals of the mean. Data shown is from experiment 1 and 3.</p

Comparison of growth and virulence of wild-type and transformed isolates of <i>Phialophora gregata</i> Type B.

a<p>Measurements taken after 21 days of growth on green bean agar.</p>b<p>Measurements taken after 21 days of growth in soybean seed broth.</p>c<p>Stem ratings are the mean of six stems 7 weeks after inoculation.</p>d<p>Resistant cultivar.</p>e<p>Susceptible cultivar.</p>f<p>Wild-type (WT).</p>g<p>Red Fluorescent Protein (RFP).</p>h<p>Experiments were replicated three times and data were combined for statistical analysis (n = 9).</p

Latent and pathogenic infection in soybean stems infected with <i>Phialophora gregata</i> tagged with GFP or RFP.

<p>Latent (2 weeks post inoculation) and pathogenic infection (8 weeks post inoculation) of stems of Bell and Corsoy 79 either individually or co-inoculated with PgA-GFP or PgB-RFP. (A) PgA-GFP infecting a vessel of the resistant cultivar Bell during latent infection. No evidence of fungal infection was observed in the susceptible cultivar Corsoy 79 infected with either PgA-GFP (B) or PgB-RFP (C) during latent infection. (D) Bell co-inoculated with PgA-GFP (green arrow) and PgB-RFP (red arrow). (E and F) A xylem vessel of Bell colonized by both PgA-GFP and PgB-RFP. (F) is enlarged in for clarity. (G–I) Corsoy 79 inoculated with both types. A, D–I 200x magnification; B, C 100x magnification.</p

Pathogenic infection in soybean stems infected with <i>Phialophora gregata</i>.

<p>Pathogenic infection by wild-type isolates of types A and B of <i>Phialophora gregata</i> as observed 8 weeks post inoculation in cross sections of stems from a susceptible soybean cultivar (Corsoy 79). (A) No infection was observed in the non-inoculated control. (B) Inoculated with Type B. (C), A necrotic vessel with no fungal structures seen inside. (D), A vessel heavily colonized by <i>P. gregata</i>. (E)A vessel with evidence of sporulation (arrow). (F), Hyphae of <i>P. gregata</i> growing between vessels, possibly via pit pairs. (G), A longitudinal section of the xylem infected by <i>P. gregata</i>. (H and I), <i>P. gregata</i> beginning to colonize the parenchyma cells that compose the pith of the stem. Images A–G and I are 200x magnification, H 400x magnification.</p

Latent infection in soybean stems infected with <i>Phialophora gregata</i>.

<p>Latent infection as observed in stem cross sections from a BSR-susceptible soybean cultivar (Corsoy 79) infected with wild-type isolates of types A and B of <i>Phialophora gregata</i>. Images A–F were captured 2 weeks post inoculation. (A) No infection was observed in the cross sections of the apex (B) or the cross section and (C) longitudinal section of the middle of the non-inoculated control plant. (D) Necrosis in the vascular system of the root, (E) longitudinal section of a root, (F) a necrotic region from D that was cropped and magnified. Image G was captured 3 weeks post inoculation and hyphae) are beginning to colonize the xylem vessels (noted by arrow). Image A is 40x magnification, B, C, and E–G are 200x magnification, D is 100x magnification.</p

Average area of vessels of stems during latent and pathogenic infection with <i>Phialophora gregata</i>.

<p>Average area (um) of vessels of each cross section two (A) and six (B) weeks after inoculation with either type A (PgA) or B (PgB) of <i>Phialophora gregata</i> for six soybean cultivars. Letters in parentheses next to cultivars indicate the source of BSR resistance or if the cultivar is susceptible (S). Treatments were compared using an ANOVA followed by a Fisher's LSD test, and bars with different letters are significantly different (P = 0.05). Error bars indicate 95% confidence intervals of the mean. Data shown is from experiment 1 and 3. Treatments for experiment 1 were replicated three times (n = 3 plants/replication). Treatments for experiment 3 were replicated three times (n = 4 plants/replication).</p

Characterization of pXV10A, a Copper Resistance Plasmid in Xanthomonas campestris pv. vesicatoria

The efficacy of copper bactericides for control of Xanthomonas campestris pv. vesicatoria in eastern Oklahoma tomato fields was evaluated. Copper bactericides did not provide adequate control, and copper-resistant (Cu(r)) strains of the pathogen were isolated. The Cu(r) genes in these strains were located on a large indigenous plasmid designated pXV10A. The host range of pXV10A was investigated; this plasmid was efficiently transferred into 8 of 11 X. campestris pathovars. However, the transfer of pXV10A to other phytopathogenic genera was not detected. DNA hybridization experiments were performed to characterize the Cu(r) genes on pXV10A. A probe containing subcloned Cu(r) genes from X. campestris pv. vesicatoria E3C5 hybridized to pXV10A; however, a subclone containing Cu(r) genes from P. syringae pv. tomato PT23 failed to hybridize to pXV10A. Further DNA hybridization experiments were performed to compare pXV10A with pXvCu plasmids, a heterogenous group of Cu(r) plasmids present in strains of X. campestris pv. vesicatoria from Florida. These studies indicated that the Cu(r) genes on pXV10A and pXvCu plasmids share nucleotide sequence homology and may have a common origin. Further experiments showed that these plasmids are distinctly different because pXV10A did not contain sequences homologous to IS476, an insertion sequence present on pXvCu plasmids