Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Summary

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Abstract

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

Introduction

Herbicides are the most extensively used weed control measure, accounting for up to 50% of the global plant protection market ¹. They are relatively cheap tools, avoid labor-intensive and time-consuming soil cultivation practices, and ultimately result in cost-effective, safe and profitable food production ². However, the great phenological and genetic variability present in many weed species, together with an over-reliance on herbicide use, frequently results in the selection of herbicide-resistant weed populations. The introduction of selective herbicides with a very specific metabolic target ^3-5 has dramatically increased the number....

Protocol

1. Seed Sampling and Storage

1. Monitor cultivated fields for unjustified poor herbicide performance, i.e., not due to adverse climatic conditions or low quality herbicide treatments.
2. Collect a seed sample from one species at a time and assign a unique code. Mature seeds are usually collected before crop harvesting from plants that had survived the herbicide treatment(s). Timely monitor to observe if the seeds are shed by mother plant when mature.
3. Fill in a form for each sample indicating the assigned unique code, name of species, collection date, GPS coordinates, municipality, farmer’s name, field size, infestation level, c....

Results

To assess the resistance status of a putative resistant population, it is fundamental to include a susceptible check in the assay in order to verify the herbicide efficacy. The results of a screening test conducted on P. rhoeas populations, a weed infesting wheat fields, are reported in Figure 2, where the efficacy of four post-emergence herbicides on a susceptible check (09-36) and on the suspected resistant one (10-91) are presented. Population 09-36 was completely controlled by the ALS inhibi.......

Discussion

Several steps within the protocols are critical for a successful assessment of herbicide resistance in a population: 1) seeds should be collected when mature from plants that had survived the herbicide treatment(s). Maturation of the seeds on the mother plant is crucial to avoid difficulties in seed germination later; 2) the proper storage of seeds is recommended to avoid proliferation of molds that would prevent germination; 3) seedlings should be treated at the right growth stage, as reported on the label of the herbic.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Paper bags	Celcar SAS
Plastic dishes	ISI plast S.p.A.	SO600	Transparent plastic
Sulfuric acid 95-98%	Sigma-Aldrich	320501
Non-woven fabric	Carretta Tessitura	Art.TNT17	Weight 17 g m-2
Chloroform >99.5%	Sigma-Aldrich	C2432
Agar	Sigma-Aldrich	A1296
Potassium nitrate >99.0%	Sigma-Aldrich	P8394
Plastic containers	Giganplast	1875/M	600 x 400 x 110 mm
Plastic trays	Piber plast	G1210A	325 x 265 x 95 mm
Polystyrene trays	Plastisavio	S24	537 x 328 x 72 mm, 24 round cells (6x4)
Copper sulfate	Sigma-Aldrich	451657
Agriperlite	Blu Agroingross sas	AGRI100
Peat	Blu Agroingross sas	TORBA250
Germination cabinet	KW	W87R
Nozzles	Teejet	XR11002-VK, TP11001-VH	The second type of nozzles are used only for glyphosate
Barcode generator	Toshiba TEC	SX4
Labels with barcode	Felga	TT20200	Stick-in labels with rounded corners
Barcode reader	Cipherlab	8300-L	Portable data terminal
Bench sprayer			Built in house
Herbicides included in the results:
Commercial product	Active ingredient	Company	Comments
Altorex	imazamox	BASF
Azimut	florasulam	Dow AgroSciences
Biopower		Bayer Crop Science	Surfact to be used with Hussar WG
Dash		BASF	Surfact to be used with Altorex
Granstar	tribenuron-methyl	Dupont
Gulliver	azimsulfuron	Dupont
Hussar WG	iodosulfuron	Bayer Crop Science
Nominee	bispyribac-Na	Bayer Crop Science
Roundup	glyphosate	Monsanto
Trend		Dupont	Surfact to be used with Granstar and Gulliver
Viper	penoxsulam	Dow AgroSciences
Weedone LV4	2,4-D	Isagro