Effects of the Fungal Bioherbicide, Alternaria cassia on Peroxidase, Pectinolytic and Proteolytic Activities in Sicklepod Seedlings

Abstract

Certain plant pathogens have demonstrated potential for use as bioherbicides for weed control, and numerous studies have been published on this subject for several decades. One of the early examples of an important fungal bioherbicide is Alternaria cassiae, isolated from the weed sicklepod (Senna obtusifolia). To gain further insight into biochemical interactions of this fungus and its host weed, we examined the effects of this bioherbicide on various enzymes associated with plant defense. Young sicklepod seedlings were challenged with A. cassiae spore inoculum and enzyme activities associated with plant defense (peroxidase, proteolytic, and pectinolytic) were assayed periodically over a 96-h time course on plants grown in continuous darkness or continuous light. Peroxidase activity increased with time in untreated control seedlings in both light and dark, but the effect was greater in the light. In A. cassiae-treated plants, peroxidase was elevated above that in control tissue at all sample times resulting in a 1.5 -fold increase above control in light-grown tissue and a 2- to 3-fold increase in dark-grown tissue over 48-96 h. Differences in leucine aminopeptidase activity in control versus A. cassiae-treated tissues were not significant until 48-96 h, when activity was inhibited in fungus-treated tissues by about 32% in light-grown tissue and 27% in dark-grown tissue after 96 h. Proteolytic activity on benzoyl-arginine-p-nitroanilide was not significantly different in treated versus control tissue in either light or dark over the time course. Pectinase activity increased in treated tissues at all time points as early as 16 h after spore application in light- or dark-grown plants. The greatest increases were 1.5-fold above control levels in light-grown plants (40-64 h) and 2-fold in plants grown in darkness (72-96 h). Data suggests that peroxidase may be involved as defense mechanism of sicklepod when challenged by A. cassia and that this mechanism is operative in young seedlings under both light and dark growth conditions. Differential proteolytic activity responses on these two substrates suggests the presence of two different enzymes. Increased pectinase activity during pathogenesis suggests that A. cassiae-sicklepod interaction results in an infectivity mechanism to degrade pectic polymers important to sicklepod cell wall integrity. These studies provide important information on some biochemical interactions that may be useful for improvements to biological weed control programs utilizing plant pathogens. Such information may also be useful in genetic selection and manipulation of pathogens for weed control.

Keywords: Alternaria cassiae; Senna obtusifolia; biological weed control; fungal bioherbicide; hydrolytic enzymes; peroxidase; plant defense; sicklepod.

Figure 1

Sicklepod seedlings produced by germinating seeds in paper toweling and grown in continuous darkness.

Figure 2

Photomicrographs of A. cassiae spores and mycelium (stained with lactophenol blue). Micrograph (a) = spores germinated on a microscope slide in soybean oil:H₂O (1:1) and (b) = spores with mycelium on sicklepod cotyledon. Arrowheads point to mycelium; arrowheads-asterisk point to spores.

Figure 3

Peroxidase activity extracted from control (un-inoculated) and A. cassiae inoculated sicklepod seedlings at various time points. Graph (a) = dark growth conditions; graph (b) = light growth conditions. Control, A. cassiae.

Figure 4

Leucine aminopeptidase activity extracted from control (un-inoculated) and A. cassiae sicklepod seedlings at various time points. Graph (a) = dark growth conditions; graph (b) = light growth conditions. Control, A. cassiae.

Figure 5

BAPA aminopeptidase activity extracted from control (un-inoculated) and A. cassiae inoculated sicklepod seedlings at various time points in seedlings grown under light growth conditions. Control, A. cassiae.

Figure 6

Pectinase activity extracted from control (un-inoculated) and A. cassiae inoculated sicklepod seedlings at various time points. Graph (a) = dark growth conditions; graph (b) = light growth conditions. Control, A. cassiae.