Modifying Myxococcus xanthus protoporphyrinogen oxidase to plant codon usage and high level of oxyfluorfen resistance in transgenic rice

Protoporphyrinogen oxidase (Protox) of Myxococcus xanthus (Mx Protox) is a 49-kDa membrane protein that catalyzes conversion of protoporphyrinogen IX (Protogen IX) into protoporphyrin IX (Proto IX). Upon heterologous expression in transgenic rice plants, Mx Protox is dually targeted into plastids and mitochondria, increasing resistance against the herbicidal Protox inhibitor oxyfluorfen. Here, we describe the chemical synthesis of the Mx Protox gene by assembling several small synthetic DNA fragments derived by ligation-PCR. Codon usage in the resulting 1416-bp gene was modified to correspond to that of the Arabidopsis Protox gene, a change that resulted in a decrease in G+C content from 71 to 49%. The modified Mx Protox gene was used to generate transgenic rice plants via Agrobacterium-mediated transformation. Integration, expression, and inheritance of the transgenes were demonstrated by Southern, Northern, and Western blot analyses. In plants transformed with the modified, low G+C-content Mx Protox gene, levels of Protox expression and enzyme activity were low compared to the levels observed for plants transformed with the native Mx Protox gene. Nonetheless, like the native gene, the modified gene conferred a high level of resistance to the herbicide oxyfluorfen in a seedling growth test.     

A tobacco plastidal transit sequence cannot override the dual targeting capacity of Myxococcus xanthus protoporphyrinogen oxidase in transgenic rice

The effect of a plastidal transit sequence in Myxococcus xanthus protoporphyrinogen oxidase (Protox) on gene targeting ability was investigated by generating transgenic rice that overexpressed M. xanthus Protox with the additional plastidal transit sequence (TTS line). In transgenic lines TTS3 and TTS4, the Protox antibody cross-reacted with the mature M. xanthus Protox protein of 50 kDa. In an in vitro import system using the M. xanthus Protox gene with the plastidal transit sequence, M. xanthus protein was detected in both chloroplasts and mitochondria, confirming that it was targeted into both organelles, as in transgenic rice line, M4, that overexpressed M. xanthus Protox lacking the plastidal transit sequence. A prominent increase in chloroplastic and mitochondrial Protox activity was observed in TTS3 and TTS4 relative to the wild type. However, the increase was lower than that in transgenic line M4. Seeds from all transgenic lines (TTS3, TTS4, and M4) were able to germinate when treated with up to 500 μM of the Protox-inhibiting herbicide, oxyfluorfen, whereas seeds from the wild type failed to germinate even when treated at levels as low as 1 μM. After foliar application of oxyfluorfen, TTS3 and TTS4 exhibited a reduced Protox activity, however, it was much greater than uninhibited Protox activity of wild type. The great increase in conductivity was followed by the great accumulation of photodynamic protoporphyrin IX only in oxyfluorfen-treated wild-type plants, not in oxyfluorfen-treated TTS lines. The presence of the plastidal transit sequence neither excludes the intrinsic ability of subcellular translocation of M. xanthus Protox nor changes herbicide resistance in TTS lines.     

Expression of human protoporphyrinogen oxidase in transgenic rice induces both a photodynamic response and oxyfluorfen resistance

A human protoporphyrinogen oxidase (Protox) coding sequence under the control of a ubiquitin promoter was introduced into rice to determine whether transgenic rice overexpressing the human Protox gene exhibits resistance against a peroxidizing herbicide. The transgenic rice lines (H3, H4, H5, H6, H9, and H10) transcribed the human Protox mRNA, whereas hybridizing RNA band was not detected in wild-type rice, indicating that the human Protox gene had been successfully transmitted into transgenic rice plants. The transgenic lines H9 and H10 showed growth retardation and light-dependent formation of necrotic lesions. Compared with wild-type rice plants, rice with a human Protox gene had increased Protox activity and content of the photosensitizer protoporphyrin IX, and reduced chlorophyll. The photosynthetic efficiency in lines H9 and H10, as indicated by F_v/F_m, was not different from that of wild type. The two transgenic lines had decreased levels of antheraxanthin, lutein, and β-carotene and similar levels of neoxanthin and violaxanthin as compared with wild-type plants. The staining activities of catalase, peroxidase, superoxide dismutase, and glutathione reductase were higher in transgenic lines than in wild type. Line H9 germinated in the presence of 20 μM oxyfluorfen, whereas 2 μM oxyfluorfen inhibited the germination of wild-type seeds. Thus, the transgenic rice plants exhibited enhanced resistance to oxyfluorfen.     

Selectivity and mode of action of carfentrazone-ethyl,a novel phenyl triazolinone herbicide

Post-emergence application of carfentrazone-ethyl at rates as low as 2·2 g ha^-1 caused greater leaf injury and growth reduction in ivyleaf morningglory (Ipomoea hederacea) and velvetleaf (Abutilon theophrasti) than in soybean (Glycine max). The herbicide was more rapidly metabolized in the crop than in the weed species, with 26·7, 54·3 and 60·6% of the parent compound remaining in soybean, ivyleaf morningglory and velvetleaf, respectively, 24 h after exposure. The free acid metabolite, carfentrazone, was present in all species and accounted for 21·2–27·4% of the total radioactivity. Unknown metabolites (R_f 0 and 0·22) were four to five times more abundant in soybean than in the weed species. Carfentrazone-ethyl induced more leakage from leaf discs from the weeds than those from soybean and the degree of injury correlated with the amount of protoporphyrin IX (Proto IX) present in the treated tissues. Both carfentrazone-ethyl and carfentrazone were potent inhibitors of protoporphyrinogen oxidase (Protox). Therefore, the selectivity of this herbicide may, at least in part, be attributed to the lower accumulation of Proto IX in soybean than in the weeds, probably because of the ability of soybean to metabolize more carfentrazone into unknown metabolites than the weeds. © 1997 SCI.     

Inhibition profile of trifludimoxazin towards PPO2 target site mutations

BACKGROUND

Target site resistance to herbicides that inhibit protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) has been described mainly in broadleaf weeds based on mutations in the gene designated protoporphyrinogen oxidase 2 (PPO2) and in one monocot weed species in protoporphyrinogen oxidase 1 (PPO1). To control PPO target site resistant weeds in future it is important to design new PPO-inhibiting herbicides that can control problematic weeds expressing mutant PPO enzymes. In this study, we assessed the efficacy of a new triazinone-type inhibitor, trifludimoxazin, to inhibit PPO2 enzymes carrying target site mutations in comparison with three widely used PPO-inhibiting herbicides.

RESULTS

Mutated Amaranthus spp. PPO2 enzymes were expressed in Escherichia coli, purified and measured biochemically for activity and inhibition kinetics, and used for complementation experiments in an E. coli hemG mutant that lacks the corresponding microbial PPO gene function. In addition, we used ectopic expression in Arabidopsis and structural PPO protein modeling to support the enzyme inhibition study. The generated data strongly suggest that trifludimoxazin is a strong inhibitor both at the enzyme level and in transgenics Arabidopsis ectopically expressing PPO2 target site mutations.

CONCLUSION

Trifludimoxazin is a potent PPO-inhibiting herbicide that inhibits various PPO2 enzymes carrying target site mutations and could be used as a chemical-based control strategy to mitigate the widespread occurrence of PPO target site resistance as well as weeds that have evolved resistance to other herbicide mode of actions. © 2022 BASF SE and The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Resistance pattern and antioxidant enzyme profiles of protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice

We quantified the resistance levels of transgenic rice plants, expressing Myxococcus xanthus protoporphyrinogen oxidase (PROTOX) in chloroplasts and mitochondria, to PROTOX inhibitors, acifluorfen, oxyfluorfen, carfentrazone-ethyl, and oxadiazon. We also determined whether active oxygen species-scavenging enzymes are involved in the resistance mechanism of transgenic rice. The transgenic rice line M4 was about >200-fold more resistant to oxyfluorfen than the wild-type (WT). M4 was also resistant to acifluorfen, carfentrazone-ethyl, and oxadiazon, but did not show multiple resistance to imazapyr and paraquat, which have different target sites. Acifluorfen, oxyfluorfen, carfentrazone-ethyl, and oxadiazon reduced the chlorophyll content in leaves of WT, but had minimal or no effect on M4. The PROTOX inhibitors also caused significant lipid peroxidation in the treated leaves of WT rice. However, the malondialdehyde production in M4 was not affected by these herbicides. The WT rice had higher activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase than M4 after treatment with PROTOX inhibitors. A similar response was observed in all cases of antioxidant isozyme profiles analyzed. However, the induction in antioxidant activity in WT was not enough to overcome the toxic effects of a PROTOX inhibitor so the plant eventually died.     

Pyrazole phenyl ether herbicides inhibit protoporphyrinogen oxidase

Two isomeric pairs of pyrazole phenyl ether herbicides [AH 2.429, 4-chloro-1-methyl-5-(4-nitrophenoxy)-3-(trifluoromethyl)-1H-pyrazole; AH 2.430, 4-chloro-1-methyl-3-(4-nitrophenoxy)-5-(trifluoromethyl)-1H-pyrazole; AH 2.431, 5-((4-chloro-1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)oxy)-2-nitrobenzoic acid; and AH 2.432, 5-((4-chloro-1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)oxy)-2-nitrobenzoic acid were evaluated for herbicidal activity in both intact plants and in tissue sections. Their capacity to induce accumulation of porphyrins in tissue sections and to inhibit protoporphyrinogen oxidase (Protox) in vitro were determined. In whole plant tests, the order of herbicidal activity was AH 2.430 AH 2.431 > AH 2.429 > AH 2.432. AH 2.430 consistently caused light-dependent membrane leakage in both green and far-red light grown cucumber cotyledon and barley primary leaf tissue sections after incubation for 20 hr in darkness in 0.1 mM solutions. The same treatment caused marked increases in protoporphyrin IX (PPIX) content during the 20-hr dark incubation. AH 2.429 and 2.431 were less effective and not effective in all tissues in causing herbicidal damage and PPIX accumulation. AH 2.432 was ineffective in tissue section assays. Mg-PPIX levels were not significantly affected by any of the compounds. Protochlorophyllide levels were decreased by AH 2.430 and 2.431 in barley and increased by AH 2.429, 2.431, and 2.432 in cucumber. A positive relationship was found between herbicidal activity and the amount of PPIX that was caused to accumulate by each compound. All of the compounds inhibited Protox activity. Positive correlations were found between herbicidal activity in planta over a 300-fold range and in vitro Protox inhibition and the amount of PPIX caused to accumulate in vivo. These data support the view that the pyrazole phenyl ethers exert their herbicidal activity entirely through inhibition of Protox.     

Development of PPO inhibitor-resistant cultures and crops

Recent progress in the development of protoporphyrinogen oxidase (PPO, Protox) inhibitor-resistant plant cell cultures and crops is reviewed, with emphasis on the molecular and cellular aspects of this topic. PPO herbicide-resistant maize plants have been reported, along with the isolation of plant PPO genes and the isolation of herbicide-resistant mutants. At the same time, PPO inhibitor-resistant rice plants have been developed by expression of the Bacillus subtilis PPO gene via targeting the gene into either chloroplast or cytoplasm. Other attempts to develop PPO herbicide-resistant plants include conventional tissue culture methods, expression of modified co-factors of the protoporphyrin IX binding subunit proteins, over-expression of wild-type plant PPO gene, and engineering of P-450 monooxygenases to degrade the PPO inhibitor.     

靶标为原卟啉原氧化酶的高效除草剂品种

原卟啉原ＩＸ氧化酶是血红素和叶绿素生物合成中的关键酶，是过氧化型除草剂的分子靶标，当植物用二苯醚类，酞酰亚胺以及一些吡啶衍生物等除草剂处理后，造成原卟啉原ＩＸ积累，膜脂质破坏，最终细胞死亡，同ＡＬＳ抑制剂一样，作用靶标为原卟啉原化酶的除草剂，具有用药量低，活性高，杀草谱广，对哺乳动物低毒，对环境影响较小等良好特性，本文介绍了作用靶标为原卟啉原氧化酶的代表性品种。     

Effect of four herbicides and two oils on leaf-cell membrane permeability*†

Leakage of electrolytes from leaf discs of treated Phaseolus Vulgaris L. plants was the main criterion used to study the effect of several chemicals on the permeability of leaf-cell membranes. Paraquat, diquat, dinoseb and oxyfluorfen (2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(Trifluoromethyl) benzene) increased leaf-cell membrane permeability after exposure for 12 h or less. An‘aromatic’oil caused a large increase in permeability at 2–5 min after treatment. Increases in electrolyte release were also correlated with release of soluble amino acids from the leaf discs but the former method was the more sensitive. Increase in cell membrane permeability was always associated with injury symptoms such as appearance of necrotic areas in leaves. Chlorpropham, linuron, sodium azide, glyphosate and 2,4-D at 10^?3M, as well as 1% X-77 surfactant and a non-phytotoxic isoparaffinic oil did not alter leaf-cell permeability at 12 h after treatment. Light was necessary for paraquat and oxyfluorfen to alter leaf cell permeability. Paraquat and oxyfluorfen caused a greater increase in leaf-cell permeability of a soybean mutant with yellow leaves as compared with the normal green leaves. With oxyfluorfen this difference in permeability was greater than with paraquat, and was associated with the appearance of severe necrotic injury symptoms in the yellow mutant; paraquat caused no injury symptoms.     

RT-PCR based detection of resistance conferred by an insensitive GS in glufosinate-resistant maize cell lines

Selection of maize (Zea mays L.) cell lines resistant to glufosinate was carried out using cell suspension cultures induced from an embryo. The cell suspension was cultured on MS liquid medium supplemented with 150 mg/l asparagine, 1 mg/l thiamine, 3 mg/l 2,4-D and 2% sucrose (pH 5.8) and treated with different concentrations of glufosinate herbicide. In vitro selection from these cell suspension cultures resulted in the identification of a maize cell line resistant to glufosinate. The resistance index of the resistant cell line was 120-fold more than the normal cells. Moreover, the resistance of the cell line remained stable for at least 6 months when kept in herbicide-free medium. Glutamine synthetase (GS) activity in the resistant cells was higher than the normal cells by about 2.30-fold at 5 days after treatment of 10⁻⁶ M glufosinate. The low sensitivity of GS in resistant cells resulted in the non-inhibition of the enzyme activity, hence, the high resistance of the cell line to the herbicide. Using RT-PCR to amplify the GS mRNA, a substitution of 12 nucleotides was observed in resistant maize cells compared to that of normal cells. Conceptual translation of this sequence shows a substitution of 10 amino acids in the GS protein sequence from Genbank database of NCBI. The GS gene sequence of the newly identified glufosinate-resistant maize cell was submitted to Genbank and was given the Accession No. AY339214. The results of this study indicate a functional role of mutation in the evolution of glufosinate-resistant plants. It also supports the view that glufosinate resistance in plants was primarily due to the alteration of GS making it less sensitive to inhibitory effect of the herbicide.     

Penoxsulam‐resistant barnyardgrass (Echinochloa crus‐galli) in rice fields in China

Barnyardgrass (Echinochloa crus‐galli) proliferation seriously threatens rice production worldwide. Whole‐plant bioassays were conducted in order to test the sensitivity to penoxsulam of 52 barnyardgrass populations and the resistance of six penoxsulam‐resistant populations to 12 other herbicides that are commonly used in rice fields. Among the 48 populations that had escaped penoxsulam control in the rice fields, 8.3% showed a very high level of resistance, 58.3% showed a high level of resistance and 10.4% showed a moderate level of resistance. Multiple resistance was confirmed in all six penoxsulam‐resistant populations that were tested further. They exhibited at least a moderate level of resistance; that is, to 6–10 of the total of 13 herbicides that was tested. Most of the six penoxsulam‐resistant populations showed at least a moderate level of resistance to bispyribac‐sodium, quinclorac, metamifop, cyhalofop‐butyl and oxadiazon, three populations held at least a moderate level of resistance to oxyfluorfen and pretilachlor, two populations also held at least a moderate level of resistance to pyrazosulfuron‐ethyl, pyribenzoxim and fenoxaprop‐P‐ethyl, but the resistance indices of the six populations to pendimethalin were all low. This study has confirmed resistance to pretilachlor and oxadiazon in weeds for the first time.     

Synthesis and herbicidal activity of novel heterocyclic protoporphyrinogen oxidase inhibitors

Protox inhibitors are applied as foliar sprays, thus causing very rapid cellular collapse and desiccation of many troublesome weeds in the presence of light. In many respects, they appear to be ideal herbicides, because they act rapidly and do not harm mammals under normal conditions. The main limitation to their widespread adoption is that few crops are naturally resistant to them. Crop tolerance has mainly been pursued with the synthesis of the cyclic imide classes containing 5- and 6-membered heterocycles, including pyrazole, pyridazine, 1,2,4-triazine, triazolinone and trifluoromethyluracil derivatives. Because of their structural novelties and biological performance, active investigations on heterocyclic protox inhibitors have been carried out in our laboratories and we have found 3-arylpyrroles to be a new class of light-activated, membrane-disrupting herbicides. They are active on both grass and broadleaf weeds at low rates. The synthesis and structure-activity relationships are presented.     

Photodynamic herbicides VIII. Mandatory requirement of light for the induction of protoporphyrin IX accumulation in acifluorfen-treated cucumber

It is shown that continuous illumination is mandatory for the induction of tetrapyrrole accumulation in acifluorfen-sodium-treated plants and for the photosensitization of tetrapyrrole-dependent photodynamic damage. At low concentrations of acifluorfen-sodium (up to 20 μM), photoporphyrin IX appears to be the major light-induced tetrapyrrole that accumulates in the treated plants. At higher concentrations of acifluorfen-sodium, monovinyl chlorophyllide a accumulates in addition to protoporphyrin IX. In the light, the development of photodynamic injury appears to be directly related to the accumulation of the light-induced tetrapyrroles. For example when acifluorfen-sodium-treated plants are returned to darkness, or are treated with tetrapyrrole biosynthesis inhibitors, tetrapyrrole accumulation and photodynamic injury come to a halt. In-vivo and in-organello studies failed, however, to support the commonly held hypothesis that the induction of tetrapyrrole accumulation in the light, in acifluorfen-sodium-treated plants, is only dependent on the inhibition of protoporphyrinogen oxidase. Indeed, when plastids capable of very high rates of tetrapyrrole biosynthesis and accumulation were incubated with δ-aminolevulinic acid and acifluorfen-sodium, either in darkness or in the light, a severe inhibition of protoporphyrin IX and total terapyrrole formation was observed. Althoung these results are compatible with the inhibition of tetrapyrrole formation by acifluorfensodium at the level of protoporphyrinogen oxidase, they indicate that, in addition to that inhibition, other cuellular processes are probably involved in the light-dependent accumulation of protoporphyrin IX in acifluorfensodium-treated plants.     

Characterization of acetolactate synthase from sulfonylurea herbicide‐resistant Schoenoplectus juncoides

Ten accessions of sulfonylurea‐resistant Schoenoplectus juncoides were collected from paddy fields in Japan. In order to characterize acetolactate synthase from sulfonylurea‐resistant S. juncoides, acetolactate synthase amino acid substitutions, whole‐plant growth inhibition and acetolactate synthase enzyme inhibition were examined. Schoenoplectus juncoides has two acetolactate synthase genes (ALS1 and ALS2). The sulfonylurea‐resistant accessions harbored amino acid substitutions at Pro197 or Trp574 in either ALS1 or ALS2 (the amino acid number is standardized to the Arabidopsis thaliana sequence). The whole plants of all the sulfonylurea‐resistant accessions showed resistance to imazosulfuron. The resistance level depended on the altered amino acid residues in acetolactate synthase. The acetolactate synthase enzyme that was partially purified from all the sulfonylurea‐resistant accessions was less sensitive to imazosulfuron, compared to the susceptible accession, suggesting that the resistance is related to the altered acetolactate synthase enzyme. In addition, the concentration–response inhibition of acetolactate synthase activity by imazosulfuron in the sulfonylurea‐resistant accessions was remarkably different with the presence of an amino acid substitution in either ALS1 or ALS2. Furthermore, the concentration–response inhibition of acetolactate synthase activity in the sulfonylurea‐resistant accessions with a P197S, P197T or W574L mutation showed a double‐sigmoid curve. The regression analysis of enzyme inhibition suggested that the abundance ratio of ALS1 to ALS2 enzymes was approximately 70:30%, with a range of ±15%. Taken together, these results suggest that the resistance of sulfonylurea‐resistant accessions of S. juncoides is related to altered acetolactate synthase in either ALS1 or ALS2, although the abundance of the altered acetolactate synthase in the plants is different among the sulfonylurea‐resistant accessions.     

Mode of bleaching phytotoxicity of herbicidal diphenylpyrrolidinones

A mode of action study of herbicidal diphenylpyrrolidinones was carried out through carotenoid analyses in intact Scenedesmus cells and by a cell‐free plant‐type phytoene desaturase assay using Escherichia coli transformants. A series of forty‐eight diphenylpyrrolidinones decreased the carotenoid content of Scenedesmus cells in the light and inhibited phytoene desaturase. The relationship between substituents at various positions and inhibition of phytoene desaturase is discussed. Using very active bleaching diphenylpyrrolidinones, a 10⁻⁵ M concentration affected neither the ζ‐carotene desaturase nor the protoporphyrinogen‐IX oxidase. Although some differences in their inhibitory activity were found between the in vivo and cell‐free assays, it is concluded that the compounds are essentially bleachers affecting carotenoid biosynthesis in plants. Enzyme kinetics studies with recombinant phytoene desaturase revealed a non‐competitive inhibition with respect to the substrate phytoene. A competition against the inhibitor was shown by the cofactor NADP⁺, suggesting an interaction of pyrrolidinones at the cofactor‐binding site of phytoene desaturase. © 2001 Society of Chemical Industry     

Effects of isoxaben on sensitive and tolerant plant cell cultures : II. Cellular alterations and inhibition of the synthesis of acid-insoluble cell wall material

The herbicide isoxaben is selectively phytotoxic to dicotyledonous plants, whereas most monocots are tolerant. We previously selected a soybean cell culture tolerant to isoxaben. Some effects of the herbicide on wild-type soybean cells, tolerant soybean cells, and wheat cells were compared. Cytological observations showed that isoxaben induced some disorganization of sensitive soybean cells, especially at the plasma membrane-cell wall interface. Tolerant soybean cells appeared normal in the presence of isoxaben. The growth of wild-type soybean cells was roughly equally sensitive to isoxaben as to dichlobenil, a cellulose synthesis inhibitor. By comparison, the selected soybean line and a wheat cell culture were less sensitive to isoxaben than to dichlobenil. Glucose incorporation into acid-insoluble cell wall material was more inhibited by isoxaben than by dichlobenil in the wild-type soybean cell culture. In the tolerant soybean cell culture, the incorporation was slightly inhibited by isoxaben, but remained sensitive to dichlobenil. In the wheat cell culture, dichlobenil was also more inhibitory but only at high concentrations. Other compounds, inhibitors of cellulose biosynthesis, of glycosylation of lipids or protein, or of cell division, either had no effect on the synthesis of acid-insoluble cell wall material or exerted apparently unspecific inhibitions. The results are consistent with isoxaben inhibiting the synthesis of a cell wall polysaccharide, which could be cellulose.     

A rapid reliable test for screening aryloxyphenoxy- propionic acid resistance within Alopecurus myosuroides and Lolium spp. populations

A reliable seedling bioassay was developed and tested for the rapid screening for resistance to aryloxyphenoxypropionic (APP) herbicides in Alopecurus myosuroides and Lolium spp. populations. It is based upon the difference in coleoptile length of resistant and susceptible A. myosuroides and Lolium seedlings, respectively, exposed to fenoxaprop-P acid and diclofop acid solution for 6 days in a plastic box. A 6 mg L⁻¹ fenoxaprop-P acid solution was selected as the best concentration for a reliable screening of resistant biotypes within A. myosuroides populations. At this concentration, coleoptile lengths of susceptible and resistant seedlings were shorter and longer than 10 mm respectively. Similarly, resistant seedlings within Lolium populations were easily detected at 10 mg L⁻¹ diclofop acid. At this concentration, coleoptile lengths of susceptible and resistant seedlings were shorter and longer than 20 mm respectively. For both populations, the coleoptile length distributions appear to discriminate between two kinds of APP-resistant biotypes (highly and slightly resistant).