Phoma medicaginis Isolate Differences Determine Disease Severity and Phytoestrogen Production in Annual Medicago spp.

Abstract

Phoma black stem and leaf spot disease of annual Medicago spp., caused by Phoma medicaginis, not only can devastate forage and seed yield, but also reduces herbage quality by inducing production of phytoestrogens, particularly coumestrol and 4'-O-methylcoumestrol, that can reduce ovulation rates of animals grazing infected forage. We determined the consequent phytoestrogen levels on three different annual Medicago species/cultivars (M. truncatula cv. Cyprus, M. polymorpha var. brevispina cv. Serena and M. murex cv. Zodiac), following inoculation with 35 isolates of P. medicaginis. Across the isolate x cultivar combinations, leaf disease incidence (%LDI), petiole disease incidence (%PDI), leaf disease severity (%LDS), petiole disease severity (%PDS), and leaf yellowing severity (%LYS) ranged up to 100, 89.4, 100, 58.1 and 61.2%, respectively. Cultivars Cyprus and Serena were most susceptible and cv. Zodiac the most resistant to P. medicaginis. Isolates WAC3653, WAC3658 and WAC4252 produced the most severe disease. Levels of phytoestrogens in stems ranged from 25 to 1995 mg/kg for coumestrol and from 0 to 418 mg/kg for 4'-O-methylcoumestrol. There was a significant positive relationship of disease incidence and severity parameters with both coumestrol and 4'-O-methylcoumestrol contents, as noted across individual cultivars, and as across the three cultivars overall where r=0.39 and 0.37 for coumestrol and 4'-O-methylcoumestrol, respectively (P<0.05). Although, cv. Serena was most susceptible to P. medicaginis and produced the highest levels of phytoestrogens in the presence of P. medicaginis, cv. Zodiac contained the highest levels of phytoestrogens in comparison with other cultivars in the absence of P. medicaginis. There was a 15-fold increase in coumestrol in cv. Serena but only a 7-fold increase in cv. Zodiac from infection of P. medicaginis. The study highlights that the intrinsic ability of a particular cultivar to produce phytoestrogens in the absence of the pathogen, and its comparative ability to produce phytoestrogens in the presence of the P. medicaginis, are both important and highly relevant to developing new annual Medicago spp. cultivars that offer improved disease resistance and better animal reproductive outcomes.

Phoma black stem severity and phytoestrogen production in annual Medicago spp. is primarily determined by interaction of cultivar and pathogen isolate

“…Control plants were also treated with 0.001% Tween 20, but in sterile distilled water. Incubation boxes were internally misted with sterile distilled water and box lids closed for 72 h postinoculation (hpi) to maintain high humidity (Ellwood et al, 2007; Omidvari et al, 2021).…”

“…Disease symptoms were recorded at 14 days postinoculation (dpi). Five different disease assessment parameters, as described by Omidvari et al (2021), were used as follows: (a) leaf disease incidence (LDI, %), by assessing the percentage of leaves per pot showing disease symptoms; (b) leaf disease severity (LDS, %), by calculating the percentage leaf area diseased per pot as a percentage of the total leaf area; (c) petiole/stem disease incidence (PDI, %), by calculating the percentage of petioles/stems per pot showing disease symptoms; (d) petiole/stem disease severity (PDS, %), by visually calculating the petiole/stem area diseased per pot as a percentage of the total petiole/stem area; and (e) leaf yellowing severity (LYS, %), by calculating the area showing yellow colour per pot as a percentage of the total leaf area. All recordings were done once, and by one person.…”

“…Omidvari et al (2021) determined the consequent phytoestrogen levels following inoculation with 35 isolates of P . medicaginis .…”

“…medicaginis isolate, ranging from 25 to 1995 mg/kg for coumestrol and from 0 to 418 mg/kg for 4′‐ O ‐methylcoumestrol. However, Omidvari et al (2021) also highlighted that the comparative intrinsic ability of each of cultivars Cyprus, Serena, and Zodiac to produce phytoestrogens in the absence as compared with the presence of the pathogen were not necessarily related. Some studies have also indicated that coumestrol content in annual Medicago spp.…”

“…Site and/or seasonal differences are two other parameters influencing the production of coumestrol, but such factors cannot be detected in controlled environment studies. Omidvari et al (2021) highlighted the critical need for further studies across a wider range of annual Medicago spp. and cultivars where improved disease resistance in conjunction with better animal reproductive outcomes are needed.…”