Interactions between Magnaporthiopsis maydis and Macrophomina phaseolina, the Causes of Wilt Diseases in Maize and Cotton

Abstract

Fungal pathogens are a significant threat to crops worldwide. The soil fungus, Magnaporthiopsis maydis, severely affects sensitive maize hybrids by causing the rapid wilting of plants at the maturity stage. Similarly, the soil fungus, Macrophomina phaseolina, develops in a variety of host plants, which leads to rot and plant mortality. The presence of both pathogens together in diseased cotton plants in Israel suggests possible interactions between them. Here, these relationships were tested in a series of experiments accompanied by real-time PCR tracking in maize and cotton. Despite the fact that neither of the pathogens was superior in a growth plate confrontation assay, their co-inoculum had a significant influence under field conditions. In maize sprouts and fully matured plants, infection by both pathogens (compared to inoculation with each of them alone) led to lesser amounts of M. maydis DNA but to increased amounts of M. phaseolina DNA levels. These results were obtained under a restricted water regime, while optimal water irrigation led to less pronounced differences. In water-stressed cotton sprouts, infection with both pathogens led to an increase in DNA amounts of each of the pathogens. Whereas the M. maydis DNA levels in the double infection remain high at the end of the season, a reduction in the amount of M. phaseolina DNA was observed. The double infection caused an increase in growth parameters in maize and cotton and decreased levels of dehydration in maize plants accompanied by an increase in yield production. Dehydration symptoms were minor in cotton under an optimal water supply. However, under a restricted water regime, the double infection abolished the harmful effect of M. phaseolina on the plants' development and yield. These findings are the first report of interactions between these two pathogens in maize and cotton, and they encourage expanding the study to additional plant hosts and examining the potential involvement of other pathogens.

Keywords: Cephalosporium maydis; Harpophora maydis; Macrophomina phaseolina; Magnaporthiopsis maydis; cotton; crop protection; fungus; late wilt; maize; real-time PCR.

Figure 1

Plate confrontation assays. The plate assay to identify interactions between Magnaporthiopsis maydis and Macrophomina phaseolina in a rich potato dextrose agar (PDA) culture media. (A) Left panel—M. maydis was seeded on both sides of the Petri dish. Center panel—M. Phaseolina was seeded at both poles of the dish. Right panel—the two fungi were seeded opposite each other, M. maydis on the left, and M. phaseolina on the right. (B) Magnification of the confrontation line between maydis (upper white hyphae) and M. phaseolina (lower dark hyphae). The two colonies formed a clear line between them. Images were taken after six days of growth at 28 °C ± 1 in the dark. Scale bar represents 6 mm.

Figure 2

qPCR diagnosis of M. maydis and M. phaseolina in maize sprouts. A full-growth season pot experiment under field conditions was conducted in the spring and summer of 2019 with a late-wilt-susceptible maize genotype prelude. The quantitative real-time polymerase chain reaction (qPCR) was used to identify the ability of the pathogens, alone or in mixed inoculation, to infect and colonize maize root tissues 37 days after sowing (DAS). Half of the treatments received reduced irrigation (water stress, upper panel, see Table 1). The control is soil taken from a nearby field with minor levels of M. maydis or M. phaseolina infestation. The y-axis values are M. maydis relative DNA (Mm) or M. phaseolina relative DNA (Mpk) abundance normalized to the cytochrome c oxidase (Cox) DNA. Vertical upper bars represent the standard error of the mean of 10 replications (pots, each containing one plant). When existing, significance from the control is indicated as * = p < 0.05.

Figure 3

qPCR diagnosis of M. maydis and M. phaseolina in maize at the end of the season. The experiment is described in Figure 2. M. maydis relative DNA (Mm) or M. phaseolina relative DNA (Mpk) abundance normalized to the cytochrome c oxidase (Cox) DNA was evaluated at 82 DAS (25 days after fertilization, DAF) in roots in the 2018 experiment (upper panels) and 79 DAS (25 DAF) in shoots in the 2019 experiment (lower panels). Vertical upper bars represent the standard error of the mean of 10 replications (pots, each containing one plant).

Figure 4

Symptoms evaluation and yield assessment in maize. Plant developmental values and yield assessment was made for the plants in the 2019 experiment described in Figure 2. Sprout emergence, 9 DAS (A), phenological stage, 23 DAS (B), plant height, 58 DAS (C), and growth and yield assessment 79 DAS and 22 DAF (D) were determined after the inoculation of the plants with M. maydis and M. phaseolina, alone or in mixed inoculation. Vertical upper bars represent the standard error of the mean of 10 replications (pots, each containing one plant from 23 DAS onwards). When existing, significance from the control (untreated) is indicated as * = p < 0.05.

Figure 5

Maize wilt assessment carried out 79 days after sowing. Wilting percentages of the 2019 experiment described in Figure 2 were determined 21 DAF. Plants were classified as “healthy” when no apparent signs of dehydration could be identified and as “symptoms” when the upper leaves’ color started to alter to light-silver and then to light-brown, rolling inward from the edges of the leaf. When the whole plant was presenting severe dehydration with light-brown color spreading to all of its parts and its cobs tilted down, it was classified as “diseased.” Percentages are the mean of 10 replications.

Figure 6

Photograph of the maize and cotton plants in the 2018 experiment. The experiment at the vegetative growth stage, 50 and 90 DAS in maize and cotton, respectively (upper panels), and at the end of the season, 82 and 120 DAF in maize and cotton, respectively (lower panels).

Figure 7

qPCR diagnosis of M. maydis and M. phaseolina in cotton sprouts. The full-growth season pot experiment under field conditions was conducted in the spring and summer of 2019. The charcoal-rot-susceptible cotton genotype Pima cotton, Goliath cv. was chosen for this experiment. The qPCR was used to identify the ability of the pathogens, alone or in mixed inoculation, to infect and colonize the cotton root tissues 37 days after sowing (DAS). The control is a soil taken from a nearby field with minor levels of M. maydis or M. phaseolina infestation. The y-axis values are M. maydis relative DNA (Mm) or M. phaseolina relative DNA (Mpk) abundance, normalized to the cytochrome c oxidase (Cox) DNA. Vertical upper bars represent the standard error of the mean of 10 replications (pots, each containing one plant).

Figure 8

qPCR diagnosis of M. maydis and M. phaseolina in cotton at the end of the season (154 DAS). The experiment was conducted in 2018. The control is a soil taken from a nearby field with minor levels of M. maydis or M. phaseolina infestation. M. maydis relative DNA (Mm) or M. phaseolina relative DNA (Mpk) abundance was normalized to the cytochrome c oxidase (Cox) DNA. Vertical upper bars represent the standard error of the mean of 10 replications (pots, each containing one plant).

Figure 9

Symptoms evaluation and yield assessment in cotton. Plant developmental values and yield assessment was made for plants in the 2019 experiment described in Figure 7. Sprout emergence (A) and height, (B) 9 DAS, phenological development, 41 DAS (C), and growth and yield assessment, 162 DAS (D), were determined after the inoculation of the plants with M. maydis and M. phaseolina, alone or in mixed inoculation. Vertical upper bars represent the standard error of the mean of 10 replications (pots, each containing one plant 41 DAS onwards). When existing, significance from the control (untreated) is indicated as * = p < 0.05.

Figure 10

Photograph of the cotton plants under a restricted water regime, in the 2019 experiment (described in Figure 7). The field was photographed 162 DAS. A decreased development of the plants in the M. phaseolina inoculation pots can be seen.