Leaves of coca are the primary source of cocaine. There is interest in using Fusarium oxysporum as a mycoherbicide in addition to other measures to control coca production, but information on coca physiology, including the stress responses of coca leaves, is limited. Deleafing coca plants stimulates rapid production of new leaves, and young expanding leaves readily abscise if treated with ethylene. Commercial preparations of cell wall degrading enzymes, as well as a 24-kDa elicitor from Fusarium oxysporum, induced significant levels of ethylene production by coca leaves. Ethylene pretreatment of coca leaves enhanced the production of ethylene by coca leaves in response to the cell wall degrading enzyme preparation, Driselase, and the 24-kDa elicitor. However, ethylene pretreatment did not enhance the rate of necrosis induced in response to either Driselase or purified 24-kDa elicitor. Driselase failed to elicit levels of necrosis comparable to the 24-kDa elicitor even at 30-fold higher protein concentrations. The response of coca leaves to the 24-kDa elicitor saturated at 6.7 μg ml−1. Age of coca leaves influenced both the level of resulting necrosis and the amount of ethylene produced in response to protein. Very young leaves produced the highest levels of ethylene and necrosis in response to Driselase and the 24-kDa elicitor. The data suggest that responsiveness of coca leaves to control measures may be synchronized over the first few weeks following defoliation.

The fungal protein Nep1, produced by Fusarium oxysporum f. sp. erythroxyli in liquid culture, caused extensive necrosis to Centaurea maculosa when water solutions of Nep1 (5 µg ml−1) and an organosilicone surfactant (1,1,1,3,5,5,5-heptamethyltrisiloxanyl propyl-methoxy-poly[ethylene oxide]) were applied as foliar sprays. Nep1 did not cause necrosis when applied with a nonionic surfactant or organosilicone surfactant plus unrefined corn oil. Plant age, protein concentration, organosilicone surfactant concentration, and the presence of a dew period influenced the amount of necrosis caused by Nep1. The addition of an 18-h dew period after treatment resulted in an increase of 10% or more in foliar necrosis at the 0.313 and 1.25 µg ml−1 (0.40 and 1.62 g ai ha−1) Nep1 concentrations. Increasing the spray volume from 129 ml m−2 (1,291.3 L ha−1) to 516 ml m−2 (5,165.2 L ha−1) more than doubled the amount of foliar necrosis caused by the 0.313 µg ml−1 (0.40 g ai ha−1 vs. 1.62 g ai ha−1) Nep1 concentration. A maximum necrosis rating of 95% was reached by 1.25 µg ml−1 Nep1 applied at 516 ml m−2 (6.46 g ai ha−1) followed by an 18-h dew period. Nep1 (6.46 g ai ha−1) remained active when coapplied to Centaurea maculosa with the herbicides 2,4-D or glyphosate (0.13 to 2.58 kg ai ha−1), causing foliar necrosis prior to the herbicides killing Centaurea maculosa. An increase in the organosilicone surfactant concentration from 1 to 2 ml ai L−1 was required to achieve levels of Nep1-induced necrosis on Centaurea maculosa acclimated to direct sun comparable to levels achieved on greenhouse-grown plants. Repeated application of Nep1 (6.48 g ai ha−1) 3 wk after an initial treatment (6.48 g ai ha−1) prevented the recovery of acclimated Centaurea maculosa. Greater damage was caused to acclimated Centaurea maculosa when Nep1 was applied near the middle of the day (80% necrosis at 10:00 A.M. and 85% necrosis at 2:00 P.M.) compared to early or late in the day (25% necrosis at 6:00 A.M. and 10% necrosis at 6:00 P.M.).

A small assortment of microbial proteins have the ability to activate defense responses and induce necrosis in plant cells through cell signaling pathways. These proteins are of interest because of their potential use as bioherbicides and inducers of plant resistance in agriculture. A 24-kDa protein (Nep1) was purified from culture filtrates of Fusarium oxysporum, and the effects of this protein on weed leaves were investigated. This protein induced necrosis in detached leaves of Papaver somniferum, Lycopersicon esculentum, Malva neglecta, and Acroptilon repens when taken up through the petiole. The pattern and level of necrosis were dependent on the plant species. Treatment with Nep1 induced the production of ethylene in isolated leaves of various species, and the level of ethylene response was shown to be correlated to the concentration of the protein. Pretreating leaves of P. somniferum, L. esculentum, M. neglecta, and Cardaria draba with 100 µl L−1 ethylene enhanced the protein induction of ethylene biosynthesis in those leaves. Application of Nep1 (200 nM) as a spray to intact plants of Abutilon theophrasti, P. somniferum, Centaurea solstitialis, Centaurea maculosa, and Sonchus oleraceus resulted in extensive necrosis of leaves within 48 h. The results of this research are supplemental to our understanding of the role of specific polypeptides in plant/microbe interactions and demonstrates for the first time that a fungal protein can cause extensive necrosis when applied to weed species as a foliar spray.

The cryptogein (Crypt) gene was obtained by PCR amplification of genomic DNA from Phytophthora cryptogea and confirmed by DNA sequencing. A promoter of the rice phenylalanine ammonia-lyase (PAL) gene was used to regulate the expression of Crypt, because this promoter has a low level of constitutive expression, is strongly induced by pathogen infection and is expressed in epidermal tissues. These promoter characteristics may be suitable for potential inhibition of pathogen attack on epidermal tissues. For functional interaction of Crypt with its outer plasma membrane binding sites, Crypt was led by a signal sequence of the extracellular pathogenesis-related protein (PR1b) of tobacco (Nicotiana tabacum). The fused gene was constructed into a binary vector and the final plasmid (CHF-PAL::Crypt) was transformed into tobacco using the Agrobacterium-mediated transformation method. Twenty-two lines of transformants were obtained from selection medium containing 100 mg/l of kanamycin. Results from PCR amplification and Southern blot analysis demonstrated that Crypt was integrated into the tobacco genome. In the assay of pathogen challenge, nearly two-thirds (68.2%) of the transgenic plants showed significantly enhanced resistance against black shank fungal (Phytophthora parasitica var. nicotianae), brown spot fungal (Alternaria alternata) and wild fire bacterial (Pseudomonas syringae pv. tabaci) diseases. This observation indicates that low-level constitutive expression of Crypt gene in tobacco could have potential use in generating broad-spectrum disease-resistant plants.

A previous study had indicated that scavengers of reactive oxygen species (ROS) delayed cell death (the hypersensitive response (HR)) triggered in epidermal cells of intact, resistant, cowpea (Vigna unguiculata (L.) Walp) leaves by the monokaryotic stage of the cowpea rust fungus (Uromyces vignae Barclay race 1). This HR had been monitored by cell autofluorescence, which occurs after protoplast collapse. In the present study, when cytoplasmic disorganization was used to monitor cell death more directly, ROS-scavengers, superoxide dismutase, catalase, horseradish peroxidase, and desferal-Mn(IV) had no effect on HR development. Cytological staining for superoxide or hydrogen peroxide generation also did not reveal the presence of ROS before or during the early stages of the HR, but did, as in the previous study, suggest a role in the autofluorescence and browning of invaded cells that occur following protoplast collapse. Staining of plant mitochondria with nitroblue tetrazolium, possibly attributable to increased dehydrogenase activity but not superoxide generation, occurred transiently around invasion hyphae (monokaryotic stage of the fungus) or haustoria (dikaryotic stage) of the fungus as they entered a cell in the susceptible or resistant cultivar. Around invasion hyphae in epidermal cells in resistant plants, this staining diminished as cytoplasmic streaming stopped, and gradually disappeared as cell death progressed. These data are consistent with other evidence that rust fungi initially negate non-specific defensive responses in both resistant and susceptible cells as part of the establishment of biotrophy. They also suggest that the HR in the cowpea–cowpea rust fungus pathosystem is not triggered by an oxidative burst.