Biotypes Of Goosegrass Research Articles

Changes in seed bank size and dormancy characteristics of the glyphosate‐resistant biotype of goosegrass (Eleusine indica[L.] Gaertn.)

A study on changes in the seed bank size of a glyphosate (N‐(phosphonomethyl)glycine)‐resistant (R) Eleusine indica biotype was carried out on a naturally occurring infestation of a young oil palm plantation. The total number of shed seeds collected was 53% of the predicted potential total number of seeds produced by the plants. Ametryn (N‐ethyl‐N′‐(methylethyl)‐6‐(methylthio)‐1,3,5‐triazine‐2, 4‐diamine) was found to be able to control the R seed population in the soil. The periodicity of seedling emergence was influenced by rainfall subsequent to the 1 month period of seed shedding. Seed burial at 2, 10 and 20 cm depths showed that 39, 33 and 79%, respectively, of viable seed persisted in the soil after 2 years. Seeds in the state of enforced dormancy played an important role in maintaining the seed population throughout the 2 year period of burial in the soil.

Germination and seedling emergence of glyphosate‐resistant and susceptible biotypes of goosegrass (Eleusine indica[L.] Gaertn.)

Effects of environmental factors on the germination and seedling emergence of glyphosate‐resistant (R) and ‐susceptible (S) biotypes of Eleusine indica (L.) Gaertn. were examined under laboratory and greenhouse conditions. The R biotype exhibited a higher germination percentage compared with the S biotype at constant temperatures of 20 and 35°C under dark conditions, and alternating temperatures of 30/25°C, and 35/25°C during a 12 h photo period. For both biotypes, germination was optimal at alternating temperatures of 30/20°C and 35/20°C. However, there was no significant difference (P > 0.05) in the germination between the R and S biotypes at these temperature regimes. The germination of both biotypes was inhibited by osmotic stress imposed by a water potential of −0.80 MPa. When the moisture stress was released and the seeds were subsequently transferred to distilled water, the germination was enhanced to approximately 90% and 16% for the R and S biotype seeds, respectively. Higher emergence rates were obtained in shallow seed depths (0 or 2 cm) compared to deep depths. Emergence percentage of the R biotype was higher than that of the S biotype at 0 cm and 2 cm depths. The maximum emergence percentage of the R biotype was higher than that of S biotype when seeds were sown on the surface of either loamy or clay loam soil taken from three different sites.

A first report of glyphosate-resistant goosegrass (Eleusine indica (L) Gaertn) in Malaysia

Goosegrass (Eleusine indica (L) Gaertn) is a noxious weed in vegetable and fruit cultivation in Malaysia. Goosegrass biotypes resistant to aryloxyphenoxypropionate and cyclohexanedione herbicides have been reported in localised areas of Malaysia since 1989. However, this weed could be easily controlled by glyphosate at 720 g isopropylamine salt ha−1. In late 1997, a fruit grower in Teluk Intan, Perak, Malaysia, reported that glyphosate failed to give adequate control of E indica in his four-year-old orchard. In response to the grower's feedback, an on-site field trial was initiated to evaluate the efficacy of glyphosate at 720 to 5760 g salt ha−1 for control of the weed. Results from this trial confirmed the grower's observation. Glyphosate at 5760 g salt ha−1 gave only about 25% control of E indica (‘Teluk Intan’ biotype). In an attempt to rule out environmental and other agronomic factors, and to further quantify the level of resistance, seeds of the biotype were collected and cultivated in our field station. In comparison to a susceptible goosegrass, the ‘Teluk Intan’ biotype was found to be 8- to 12-fold resistant to glyphosate. © 2000 Society of Chemical Industry

Inheritance of resistance to anti‐microtubule dinitroaniline herbicides in an “intermediate” resistant biotype of Eleusine indica (Poaceae)

Inheritance of resistance to the anti-microtubule dinitroaniline herbicides was investigated in a goosegrass biotype displaying an intermediate level of resistance (I). Reciprocal crosses were made between the I biotype and previously characterized susceptible (S) or resistant (R) biotypes. Eight F(1) hybrids were identified, and F(2) populations were produced by selfing. The dinitroaniline-herbicide response phenotype (DRP) of F(1) plants, and F(2) seedlings was determined using a root-growth bioassay. The DRP of F(1) plants of S × I was "susceptible" (i.e., identical to the S parental plants), and the DRP of F(1) plants of I × R was "intermediate" (i.e., identical to the I parental plants). Nonparental phenotypes were not observed in F(1) plants. Results indicated susceptibility to be dominant over intermediate resistance and intermediate resistance to be dominant over high resistance. Analysis of reciprocal crosses ruled out any role for cytoplasmic inheritance. When treated at the discriminating concentration (e.g., 0.28 ppm oryzalin), F(2) seedlings of S × I were classified as either S or I phenotype, and F(2) seedlings of I × R were classified as either I or R phenotype. Again, nonparental phenotypes were not observed. The 3:1 (S:I or I:R) segregation ratios in F(2) seedlings were consistent across all eight F(2) families. The results show that dinitroaniline herbicide resistance in the I biotype of goosegrass is inherited as a single, nuclear gene. Furthermore, it suggests that dinitroaniline resistance in goosegrass is controlled by three alleles at a single locus (i.e., Drp-S, Drp-i, and Drp-r).

Molecular characterization of four beta-tubulin genes from dinitroaniline susceptible and resistant biotypes of Eleusine indica.

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, resistant biotypes of goosegrass (Eleusine indica) developed in previously susceptible wild-type populations. We have previously reported that alpha-tubulin missense mutations correlate with dinitroaniline response phenotypes (Drp) (Plant Cell 10: 297-308, 1998). In order to ascertain associations of other tubulins with dinitroaniline resistance, four beta-tubulin cDNA classes (designated TUB1, TUB2, TUB3, and TUB4) were isolated from dinitroaniline-susceptible and -resistant biotypes. Sequence analysis of the four beta-tubulin cDNA classes identified no missense mutations. Identified nucleotide substitutions did not result in amino acid replacements. These results suggest that the molecular basis of dinitroaniline resistance in goosegrass differs from those of colchicine/dinitroaniline cross-resistant Chlamydomonas reinhardtii and benzimidazole-resistant fungi and yeast. Expression of the four beta-tubulins was highest in inflorescences. This is in contrast to alpha-tubulin TUA1 that is expressed predominantly in roots. Collectively, these results imply that beta-tubulin genes are not associated with dinitroaniline resistance in goosegrass. Phylogenetic analysis of the four beta-tubulins, together with three alpha-tubulins, suggests that the resistant biotype developed independently in multiple locations rather than spreading from one location.

Herbicide resistance caused by spontaneous mutation of the cytoskeletal protein tubulin.

The dinitroaniline herbicides (such as trifluralin and oryzalin) have been developed for the selective control of weeds in arable crops. However, prolonged use of these chemicals has resulted in the selection of resistant biotypes of goosegrass, a major weed. These herbicides bind to the plant tubulin protein but not to mammalian tubulin. Here we show that the major alpha-tubulin gene of the resistant biotype has three base changes within the coding sequence. These base changes swap cytosine and thymine, most likely as the result of the spontaneous deamination of methylated cytosine. One of these base changes causes an amino-acid change in the protein: normal threonine at position 239 is changed to isoleucine. This position is close to the site of interaction between tubulin dimers in the microtubule protofilament. We show that the mutated gene is the cause of the herbicide resistance by using it to transform maize and confer resistance to dinitroaniline herbicides. Our results provide a molecular explanation for the resistance of goosegrass to dinitroanaline herbicides, a phenomenon that has arisen, and been selected for, as a result of repeated exposure to this class of herbicide.

Alpha-tubulin missense mutations correlate with antimicrotubule drug resistance in Eleusine indica.

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, highly resistant and intermediately resistant biotypes of goosegrass (Eleusine indica) developed in previously wild-type populations. Three alpha-tubulin cDNA classes (designated TUA1, TUA2, and TUA3) were isolated from each biotype. Nucleotide differences between the susceptible and the resistant (R) alpha-tubulins were identified in TUA1 and TUA2. The most significant differences were missense mutations that occurred in TUA1 of the R and intermediately resistant (I) biotypes. Such mutations convert Thr-239 to Ile in the R biotype and Met-268 to Thr in the I biotype. These amino acid substitutions alter hydrophobicity; therefore, they may alter the dinitroaniline binding property of the protein. These mutations were correlated with the dinitroaniline response phenotypes (Drp). Plants homozygous for susceptibility possessed the wild-type TUA1 allele; plants homozygous for resistance possessed the mutant tua1 allele; and plants heterozygous for susceptibility possessed both wild-type and mutant alleles. Thus, we conclude that TUA1 is at the Drp locus. Using polymerase chain reaction primer-introduced restriction analysis, we demonstrated that goosegrass genomic DNA can be diagnosed for Drp alleles. Although not direct proof, these results suggest that a mutation in an alpha-tubulin gene confers resistance to dinitroanilines in goosegrass.

Growth Characteristics of Selected Dinitroaniline-Resistant and -Susceptible Goosegrass (Eleusine indica) Population

Growth and development of selected dinitroaniline-resistant (DR) and -susceptible (DS) goosegrass populations were compared in field studies in replacement series, in association with cotton, and under noncompetitive conditions. The DR ‘Florence’ (FR) and DS ‘Orangeburg’ (OS) populations were similar in competitiveness as indicated by relative yields (RY) and relative crowding coefficients (RCC) for vegetative and reproductive biomass production in a replacement series. Relative growth of the FR and DS ‘Anderson’ (AS) populations in replacement series was similar when vegetative biomass was considered, but reproductive biomass of the AS population declined as proportion of FR population increased. RCC values also indicated that the FR population interfered with reproductive development of the AS population. In field studies, the FR population produced greater vegetative biomass than either DS population, while similar reproductive biomass occurred for the FR and OS populations. Inflorescence dry weights were greater for the FR than the AS population under noncompetitive conditions and in cotton, but were greater for the AS population in replacement series studies. The competitiveness of DR and DS goosegrass biotypes appears to be more related to growth characteristics of individual populations than to response to dinitroaniline herbicides.

Molecular Characterization of the Tubulin-Related Gene Families in Herbicide Resistant and Susceptible Goosegrass (Eleusine indica)

Goosegrass, wide spread throughout the tropics and subtropics, is one of the most noxious weeds known. Recently, biotypes of goosegrass have been found resistant to the dinitroaniline herbicides. An alteration in the structure/composition of a tubulin protein has been postulated as an explanation for the hyperstable microtubules and the resistant phenotype. Our study was initiated to investigate the structure of the alpha (α)-, beta (β)- and gamma (γ)-tubulin related gene sequences in resistant, intermediately resistant, and susceptible biotypes. Heterologous tubulin gene clones were used as probes of restriction endonuclease-digested genomic DNA from each biotype, to determine gene size and copy number and to screen for restriction fragment length polymorphisms. The tubulin genes are organized into gene families. There are three to five α-tubulin genes, four to seven β-tubulin genes, and four to eight γ-tubulin genes. There was no evidence of multiple copies or tandem repeats of any individual gene sequence. Although RFLPs were observed, no significant difference in the banding pattern between the resistant and the susceptible biotypes was found for either α-, β-, or γ-tubulin gene families. Therefore, it is unlikely that the difference between the herbicide-response phenotypes can be attributed to large deletions or insertions in a tubulin gene.

MOLECULAR CHARACTERIZATION OF THE BETA-TUBULIN GENE FAMILY IN HERBICIDE-RESISTANT AND -SUSCEPTIBLE GOOSEGRASS [ELEUSINE INDICA (L.) GAERTN.

Goosegrass is a noxious weed in cotton fields as well as in other agronomic and horticultural crops. Normally this weed has been effectively controlled by the pre-emergent dinitroaniline herbicides. Recently a dinitroaniline resistant biotype of goosegrass has been reported from cotton fields in the southeastern U.S.(Mudge et ai., 1984, Weed Science 32 : 591-94). At the subcellular level, the dinitroanilines appear to affect the stability of the microtubules by altering the polymerization/depolymerization cycle of the tubulin monomers. Therefore this herbicide would deleteriously affect vital cellular functions, such as nuclear and cell division in susceptible plants. There is a question regarding the presence and role of an altered beta-tubulin in the herbicide response phenotype expressed by the resistant biotype of goosegrass (Vaughn et al., 1987, Plant Physiol 83 : 956-64 ; Waldin et ai., 1992, Planta 188 : 256-64). We have undertaken an analysis of the beta-tubulin gene(s) in goosegrass to clarify the role of beta tubulin in the herbicide response phenotype. Initial results, from Southern blot analysis using heterologous gene probes, indicate 4 to 6 members in the gene family. We will report the results of our on going studies to characterize the size, structure and complexity of the beta-tubulin multigene family in goosegrass.

GENETIC ANALYSIS OF DINITROANILINE-RESISTANT AND SUSCEPTIBLE GOOSEGRASS [ELEUSINE INDICA (L.) GAERTN.j.

Goosegrass biotypes resistant to the dinitroaniline herbicides have recently been identified in many of the cotton growing areas of the southeastern U.S. Knowledge of the molecular basis of this resistance will be critical to understanding the genetics and evolution of this interesting and agronomically important phenotype. Our initial efforts have focused on developing bioassays (e.g., root and pollen tube growth) and identifying genetic markers (using isoenzymes, RFLP's and RAPD's) for these herbicide-response phenotypes. Although 7 of 15 isozyme systems were polymorphic in our study populations, and 63% of the among-population variance could be attributed to population location, none of the variance could be attributed to differences in herbicide response. We have identified 8 RAPD markers that are, at least, population specific. Four of these appear to be herbicide-response specific in our initial evaluations. We have initiated an RFLP analysis using heterologous, ß-tubulin gene probes. The genetic markers will be used to identify hybrid progeny of a cross between inbred lines of resistant and susceptible biotypes, in order to determine the number of genes involved in the expression of resistance to the dinitroanilines.

Cellular Characteristics of Dinitroaniline Herbicide-Resistant Goosegrass (Eleusine indica)

Primary root cells from five dinitroaniline-resistant (R) and three susceptible (S) goosegrass biotypes from North Carolina and South Carolina were observed by transmission electron microscopy to determine whether resistance was associated with changes in cell wall formation. Cell wall malformations were found in some cells from two of the R-biotypes and in one of the S-biotypes. Malformations consisted of partially deposited cell walls and the inclusion of cell wall material in the cytoplasm. Some of the affected cells also had abnormal, lobed nuclei and malformed mitochondria. There seems to be little or no correlation between dinitroaniline resistance and cell wall malformations.

A Biotype of Goosegrass (Eleusine indica) With an Intermediate Level of Dinitroaniline Herbicide Resistance

Two kinds of dinitroaniline-resistant biotypes of goosegrass have been found in counties in South Carolina where trifluralin is used for weed control. One biotype, termed the resistant (R) biotype, resists trifluralin about 1000- to 10 000-fold. The other biotype is intermediate (I) in resistance between the R and susceptible (S) biotypes, resisting trifluralin about 50-fold. Growth studies and root tip squashes were used to determine the level of resistance to other dinitroaniline herbicides. The R biotype is cross resistant to all dinitroaniline herbicides, and the I biotype is highly resistant to some and is resistant to others only at a low level. The cross resistance of the I biotype to other dinitroaniline herbicides does not correlate with structural or solubility characteristics of the herbicides. Electron microscopic investigations indicate that cell plate formation (controlled by phragmoplast microtubule arrays) is affected at lower herbicide concentrations than spindle microtubule formations. The level of herbicide resistance, the pattern of cross resistance, and the ultrastructural effects induced by the herbicide indicate that the I biotype has a unique mechanism of resistance from the R biotype.

Effects of Trifluralin Metabolites on Goosegrass (Eleusine indica) Root Meristems

The effects of treatment with twelve metabolites of trifluralin [2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl) benzenamine] on the gross morphology and ultrastructure of trifluralin-susceptible (S) and -resistant (R) biotypes of goosegrass [Eleusine indica(L.) Gaertner. # ELEIN] root meristems were examined. Unlike trifluralin, ten of the twelve metabolites including the major metabolites of trifluralin caused no root tip swelling even at water-saturated concentrations. Two of the minor metabolites, 3-methoxy-2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine (TR-36M) andN-(2,6-dinitro-4-(trifluoromethyl)phenyl)-N-propylpropanamide (TR-40), caused root swelling when applied as a saturated solution. The most effective metabolite, TR-36M, caused root swelling at 10μM or greater concentrations. Because trifluralin caused swelling of the root tips even at a 0.01 μM concentration, trifluralin is 1000-fold more effective than the most herbicidally active metabolite. Both herbicidally active metabolites are also much less effective than trifluralin at increasing the mitotic index. Structural studies indicate that the two herbicidally active metabolites arrest mitosis at prometaphase due to the loss of spindle microtubules, similar to trifluralin. Cortical microtubules are also affected by the highest concentrations of these two metabolites. A resistant biotype, in which mitosis is unaffected by trifluralin, was unaffected by any of the trifluralin metabolites.

Resistance of Goosegrass (Eleusine indica) to Dinitroaniline Herbicides

The presence of a dinitroaniline-resistant biotype of goosegrass [Eleusine indica(L.) Gaertner. ♯3ELEIN] was confirmed in the northeastern area of South Carolina. It was also found in seven adjoining counties. It showed resistance to seven dinitroaniline herbicides but not to herbicides from six other chemical groups. One biotype was not affected by trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) at 3.4 kg ai/ha where a dinitroaniline-susceptible biotype was controlled at 0.3 kg/ha. Under similar conditions, another biotype showed resistance to trifluralin at 0.6 kg/ha but was controlled at 1.1 kg/ha, indicating that at least two unique biotypes may exist.