Plants of Sumatran fleabane [Conyza sumatrensis (Retz.) E. Walker] were identified in a field with an unusual rapid leaf-injury herbicide symptoms after application of 2,4-D in mixture with glyphosate. The objectives of this study were to confirm the occurrence of resistance to 2,4-D herbicide and to characterize the occurrence of rapid necrosis as the mechanism associated with the herbicide resistance in C. sumatrensis. The studies performed were an initial screening, effect of 2,4-D alone and associated with glyphosate, cross- and multiple-resistance evaluation, effect of commercial formulation and analytical product, and rate of H2O2 evolution. The Marpr9-rn accession was identified with rapid necrosis symptoms and survival to 804 g ae ha−1 of 2,4-D. The resistance factor to the herbicide 2,4-D was 18.6 at 49 d after spraying. The analytical product 2,4-D and the commercial formulation resulted in similar symptoms of rapid necrosis. This symptom did not occur for the six other auxinic herbicides (dicamba, florpyrauxifen-benzyl, fluroxypyr, halauxifen-methyl, picloram, and triclopyr), indicating absence of cross-resistance. Multiple resistance to the herbicides paraquat, saflufenacil, and ammonium glufosinate was not identified in the Marpr9-rn population. However, survival following treatment with the herbicides glyphosate and chlorimuron-ethyl occurred. The evolution of H2O2 began at 15 min after application and was less pronounced in low light. These results indicate the first case of resistance to 2,4-D and occurrence of rapid necrosis in C. sumatrensis.

We are studying the possibility of altering the virulence and host range of a phytopathogen by transferring and expressing certain genes from the soil-dwelling saprophyte, Streptomyces hygroscopicus, in a plant pathogen model, Xanthomonas campestris pv. campestris (XCC). The genes, referred to herein as the “bialaphos genes,” encode the production of bialaphos, a potent glutamine-synthetase-inhibiting herbicide. This cluster of genes was originally isolated from several biosynthetically blocked mutants of S. hygroscopicus and constructed into a plasmid vector, pBG9. We have transferred a fragment of the gene cluster into pLAFR3, a plasmid that functions in both Escherichia coli and XCC and contains a tetracycline resistance marker. The resulting plasmid, named pIL-1, was used to transform E. coli and was incorporated into XCC by conjugation. The transfer of the fragment was confirmed by Southern analysis. The genes were maintained in XCC for about 47 generations in the absence of selection for tetracycline, and no changes in cultural phenotypes were seen in the transformed XCC (XCC/pIL-1). The XCC/pIL-1 cells were pathogenic to their natural hosts cabbage and broccoli, but induced an altered hypersensitive response in the nonhosts bean, pepper, sunflower, and tobacco. The pathogenic host-reaction, induced by the parent XCC, XCC/pLAFR3, and XCC/pIL-1, was a typical black rot disease in inoculated leaves of the two hosts. The nonhost reaction on the nonhost leaves was necrotic hypersensitivity, induced by XCC and XCC/pLAFR3, or the inhibition of hypersensitivity accompanied by only chlorosis at sites inoculated with XCC/pIL-1. We hypothesize that the altered hypersensitivity phenotype may be due to the transformed XCC becoming more compatible with the nonhosts, a step toward acquiring nonhost-virulence, or due to disruption of the normal expression of the hypersensitivity and pathogenicity genes in the transformed XCC. More work is needed to confirm that the introduced genes are being expressed in XCC. With further understanding, this approach may provide a useful model to study host range, virulence, and strain improvement of plant pathogens for biological control of weeds.

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.).

The short-term polyamine response to inoculation, with tobacco mosaic virus (TMV), of TMV-inoculated NN (hypersensitive) and nn (susceptible) plants of Nicotiana tabacum (L.) cv. Samsun was investigated. Free and conjugated polyamine concentrations, putrescine biosynthesis, evaluated through arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities, and putrescine oxidation, via diamine oxidase (DAO) activity, were analysed during the first 24 h from inoculation. Results were compared with those of mock-inoculated control plants. In NN TMV-inoculated plants undergoing the hypersensitive response (HR), free putrescine and spermidine concentrations had increased after 5 h compared with controls; polyamine conjugates also tended to increase compared with controls. In both virus- and mock-inoculated plants, ADC and ODC activities generally increased whereas DAO activity, which was present in controls, was detectable only in traces in inoculated tissues.

In TMV-infected susceptible plants, free putrescine and spermidine concentrations were lower at 5 h relative to controls, as were polyamine conjugates. No differences were revealed in ADC and ODC activities whereas DAO activity was not detectable. These results further support the hypothesis that polyamines are involved in the response of tobacco to TMV and that, only a few hours after inoculation, the response of hypersensitive plants is distinct from that of susceptible ones.

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.

Living epidermal cells of cowpea (Vigna unguiculata (L.) Walp.) were examined by contrast-enhanced video microscopy during penetration by invasion hyphae of the monokaryotic stage of the cowpea rust fungus (Uromyces vignae Barclay race 1). In resistant or susceptible host cvs, the plant nucleus migrated to the penetration site before the fungus had fully penetrated the plant wall and, at sites of unsuccessful infection, remained during the formation of a callose-containing papilla. Nuclear migration was also induced by applying hemicellulase, but not H2O2, to localized sites of wall damage. Hemicellulase-induced migration was inhibited by calcium chelators and a protein kinase inhibitor, but not catalase. In both resistant and susceptible cvs, the plant nucleus migrated away from successful infection sites at about the time that the fungal penetration peg made contact with the plant plasma membrane, and the epidermal cell showed no further cytological responses to the growth of the fungal intra-epidermal vesicle. In the susceptible cv., the nucleus migrated back to the fungus when the latter initiated tip growth. Inhibitors of transcription or translation did not affect this migration and only slightly reduced fungal growth. In the resistant cv. in which the invaded cell exhibited a hypersensitive response (HR), the plant nucleus changed its appearance before the cessation of cytoplasmic streaming and usually did not migrate to the fungus, even if the latter initiated tip growth. Nuclear DNA cleavage usually followed the subsequent cessation of cytoplasmic streaming. Treatments that delayed cell death and increased fungal growth also increased the frequency of nuclear migration to the fungus. It is argued that these and other data suggest that U. vignae negates nonspecific, penetration-induced, defence responses upon entering cells of both susceptible and resistant cultivars. The results also suggest that effects on the plant nucleus are one of the earliest signs of the HR in this system, often preceding the cessation of cytoplasmic streaming and detectable changes in plasma membrane permeability.