BACKGROUND: Resistance to protoporphyrinogen oxidase (PPO)-inhibiting herbicides is endowed primarily by target-site mutations at the PPX2 gene that compromise binding of the herbicide to the catalytic domain. In Amaranthus spp. PPX2, the most prevalent target mutations are deletion of the G210 codon, and the R128G and G339A substitutions. These mutations strongly affect the dynamic of the PPO2 binding pocket, resulting in reduced affinity with the ligand. Here we investigated the likelihood of co-occurrence of the most widespread target site mutations in the same PPX2 allele. RESULTS: Plants carrying R128G+/+ ΔG210+/−, where + indicates presence of the mutation, were crossed with each other. The PPX2 of the offspring was subjected to pyrosequencing and E. coli-based Sanger sequencing to determine mutation frequencies and allele co-occurrence. The data show that R128G ΔG210 can occur in one allele only; the second allele carries only one mutation. Double mutation in both alleles is less likely because of significant loss of enzyme activity. The segregation of offspring populations derived from a cross between heterozygous plants carrying ΔG210 G399A also showed no co-occurrence in the same allele. The offspring exhibited the expected mutation distribution patterns with few exceptions. CONCLUSIONS: Homozygous double-mutants are not physiologically viable. Double-mutant plants can only exist in a heterozygous state. Alternatively, if two mutations are detected in one plant, each mutation would occur in a separate allele.

Main conclusion Amplification and overexpression of the target site glutamine synthetase, specifically the plastid-located isoform, confers resistance to glufosinate in Amaranthus palmeri. This mechanism is novel among glufosinate-resistant weeds. Abstract Amaranthus palmeri has recently evolved resistance to glufosinate herbicide. Several A. palmeri populations from Missouri and Mississippi, U.S.A. had survivors when sprayed with glufosinate-ammonium (GFA, 657 g ha−1). One population, MO#2 (fourfold resistant) and its progeny (sixfold resistant), were used to study the resistance mechanism, focusing on the herbicide target glutamine synthetase (GS). We identified four GS genes in A. palmeri; three were transcribed: one coding for the plastidic protein (GS2) and two coding for cytoplasmic isoforms (GS1.1 and GS1.2). These isoforms did not contain mutations associated with resistance. The 17 glufosinate survivors studied showed up to 21-fold increase in GS2 copies. GS2 was expressed up to 190-fold among glufosinate survivors. GS1.1 was overexpressed > twofold in only 3 of 17, and GS1.2 in 2 of 17 survivors. GS inhibition by GFA causes ammonia accumulation in susceptible plants. Ammonia level was analyzed in 12 F1 plants. GS2 expression was negatively correlated with ammonia level (r =  – 0.712); therefore, plants with higher GS2 expression are less sensitive to GFA. The operating efficiency of photosystem II (ϕPSII) of Nicotiana benthamiana overexpressing GS2 was four times less inhibited by GFA compared to control plants. Therefore, increased copy and overexpression of GS2 confer resistance to GFA in A. palmeri (or other plants). We present novel understanding of the role of GS2 in resistance evolution to glufosinate.

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Fusarium oxysporum isolates (Foxy 2 and PSM197) are potential, highly host specific mycoherbicides for the control of the parasitic weeds Striga hermonthica and S. asiatica. Their target weeds, Striga spp., are major biotic constraints in cereal and legume production in semi-arid tropical Africa, where they adversely affect livelihood of millions of subsistence farmers. The aim of this study was to characterize and sequence the Striga mycoherbicides Foxy 2 & PSM197 in order to more clearly distinguish them from other morphologically similar pathogenic Fusarium oxysporum strains. The fungal isolates were cultivated on PDA medium and characterized based on the analysis of partial DNA sequence of the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene. Both isolates were identical in their ITSsequence. The unique and identical ITS-sequence of the two isolates obtained, compared to the sequences of Fusarium oxysporum forma speciales deposited in GenBank along with the host specificity to Striga demonstrated in previous studies, provides strong evidence to propose these pathogens of Striga as a new forma specialis (f. sp. strigae). The possibility to clearly distinguish between the new forma specialis and all pathogenic Fusarium oxysporum strains sequenced so far will facilitate and encourage the acceptance and introduction of Striga-mycoherbicides for practical field application by regulatory authorities and farmers.