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Proposed Rule

Amendments to the Pale Cyst Nematode Regulations

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Animal and Plant Health Inspection Service, USDA.


Proposed rule; reopening of comment period.


We are reopening the comment period for our proposed rule that would amend the domestic quarantine regulations for pale cyst nematode by adding procedures to allow persons to review and comment on the protocols for regulating and deregulating infested and associated areas. We are taking this action to allow persons to comment on the science on which we have established our infested and associated field protocols and on the sources we have used to develop the protocol principles and methods currently used. This action will allow interested persons additional time to prepare and submit comments.


The comment period for the proposed rule published on March 4, 2019 (84 FR 7304-7306), is reopened. We will consider all comments that we receive on or before July 6, 2020.


You may submit comments by either of the following methods:

Supporting documents and any comments we receive on this docket may be viewed at​#!docketDetail;​D=​APHIS-2018-0041 or in our reading room, which is located in Room 1141 of the USDA South Building, 14th Street and Independence Avenue SW, Washington, DC. Normal reading room hours are 8 a.m. to 4:30 p.m., Monday through Friday, except holidays. To be sure someone is there to help you, please call (202) 799-7039 before coming.

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Ms. Lynn Evans-Goldner, National Policy Manager, Office of the Deputy Administrator, PPQ, APHIS, 4700 River Road, Unit 137, Riverdale, MD 20737; (301) 851-2286;

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On March 4, 2019, we published in the Federal Register (84 FR 7304-7306, Docket No. APHIS-2018-0041) a proposal [1] to amend the domestic quarantine regulations for Globodera pallida (pale cyst nematode, or PCN) by adding procedures that allow persons to review and comment on the protocols for regulating and deregulating quarantined and associated areas. We took this action in response to a court order [2] requiring the Animal and Plant Health Inspection Service (APHIS) to solicit public input into the development of the protocols used for deregulating fields for PCN.

We solicited comments concerning our proposal for 60 days ending May 3, 2019. We reopened the comment period for 30 days ending July 26, 2019, in response to commenters who experienced technical difficulties with accessing the protocols online.

During the comment period, we made available for comment six documents: The Infested Field Confirmatory Policy, the Regulated Field Survey and Laboratory Result Definitions, the Infested Field Deregulation Protocol (if remaining in host crop production), the Associated Field Deregulation Protocol (if remaining in host crop production), the Deregulation Protocol for Agricultural Land No Longer in Host Crop Production, and the Analysis in Support of Certification that the Rule will not have a Significant Economic Impact on a Substantial Number of Small Entities.

We received a total of 19 comments, 2 of which were submitted twice. One person commented that we did not adequately explain the science and sources for our confirmatory and deregulatory field protocols contained in the applicable documents. Out of an abundance of caution and transparency, and in deference to the court which directed us to provide “requisite public notice and commenting on the Deregulation Protocols,” APHIS is providing the public with an additional opportunity to comment on the science supporting the protocols, including the sources of the methods informing their content. Accordingly, we are including more information about the protocols in this document and are reopening the comment period for 30 days.

APHIS' prompt response to finding PCN in Idaho, which resulted in the drafting and publication of the interim rule in 2007,[3] drew extensively upon the U.S. Department of Agriculture (USDA) Emergency Programs Manual (EPM) (February 2002).[4] The EPM lays out in general form the procedures necessary for addressing plant pest emergencies, including development of an interim rule that establishes survey activities, quarantines, movement restrictions, and other pest measures intended to mitigate or eradicate the pest. APHIS has implemented similar plant pest responses throughout the United States in other programs to address golden nematode, spotted lanternfly, potato wart, gypsy moth, and fruit flies. Similar types of early detection and rapid response efforts are employed by other Federal, State, and international plant protection organizations.

Based on the initial regulations for controlling PCN that we finalized through rulemaking, we subsequently developed protocols for regulating and deregulating PCN-infested and associated fields.[5] APHIS has harmonized its regulations and enforcement efforts with those of the Idaho State Department of Agriculture Start Printed Page 34538and the Canadian Food Inspection Agency. The protocol mitigations work collectively as a systems approach and have significantly reduced the rate of PCN spread by regulating infested and associated fields and establishing sanitation requirements for equipment and vehicles leaving infested and associated fields. In the absence of such regulatory measures, we note that statistical analysis of human-assisted spread of PCN estimates a mean spread rate of 3.29 miles/year.[6] This suggests that in the 14 years since PCN was first detected in Idaho, the pest could have spread more than 46 miles from the first infested field identified. With regulatory controls in place, PCN is limited to an area within an 8.5-mile radius, only 11.5 miles in straight line distance.

Below, we list the procedures used in the protocols and explain the scientific rationale and background we relied upon as grounds for including them. As noted above, many, if not most, of these procedures have been employed by USDA and State pest programs for decades across the United States, in various forms and for many different plant pests and crops, including nematodes on potatoes. Internationally, Australia and Japan, which also do not have widespread PCN infestations, have also relied on these and similar best practices to help them respond to PCN detections in their respective countries.[7]

Containment Measures for PCN

Different types of farming equipment can spread Globodera cysts,[8] with potato diggers representing the greatest potential risk. The risk is high because of the large amount of soil that adheres to the digger and because PCN population densities are highest at harvest time following production of a susceptible cultivar. Additionally, the new cysts present at harvest contain a large number of viable eggs that provide a greater chance of successful population establishment.[9] Consequently, every precaution should be taken to prevent the spread of potato cyst nematodes. Nematologists advise those who work in the fields to clean equipment of soil before entering non-infested sites.[10]

Based on these established best practices, the PCN program protocols include requirements for pressure washing or using steam to clean all farm equipment, vehicles, or other conveyances that have been in a PCN infested or associated field. These procedures ensure that nematodes are not carried into new fields via soil or equipment. Washing and steam sterilization of equipment has been a phytosanitary standard for nematode and other plant pest control for decades, and the techniques required in the PCN deregulation protocols are similar to plant pest sanitation protocols used throughout the United States and the world. More specifically, the PCN sanitation practices are modeled in part after those employed by the USDA Golden Nematode program for controlling the spread of that pest in New York State. A 2006 version of the USDA Golden Nematode Manual requires that all soil be removed by cleaning farm equipment, mechanized soil moving equipment, farm tools, used containers, and other similar articles using pressure washing and steam treatment.[11]

Soil Sampling and Detection Strategies for PCN

Soil sampling rates used by the PCN program for associated and infested fields are supported by a model that combines the medium scale distribution of cysts and the small scale distribution of cysts within square meters. The medium scale distribution provides the expected population densities at each position within the focus and refers to the size and shape of a focus resulting from farming practices. The small scale distribution represents the multiplication of Globodera on the roots of evenly spaced potato plants.

A computer program, SAMPLE, analyzes soil sampling methods.[12] The parameters of the model include gradient length and width, which represent the medium scale distribution and the aggregation factor of the negative binomial distribution (small scale distribution). Terms of the soil sampling method are also factored into the program. The terms are maximum grid cell size, sampling points per hectare (ha), core size cubic centimeters (cc), soil sample size (cc) per ha, and bulk sample size (gram). In this program, the selected average detection probability is set at 90 percent. The following sampling rates were calculated to detect extremely small infestations at three critical phases of the program: Deregulation of associated fields, monitoring eradication progress on infested fields, and deregulation of infested fields (in-field bioassay). The Canadian and United States Guidelines on Surveillance and Phytosanitary Actions for the potato cyst nematodes Globodera rostochiensis and Globodera pallida recommend a minimum sample size of 20,000 cc per ha (approximately 8,000 cc per acre) taken either manually or mechanically. When a similar method was analyzed with the SAMPLE program using 15,000 cc/ha (approximately 6,000 cc per acre) with a bulk sample size of 22.5 kilogram (kg), it had a detection probability of 99 percent with a central population density (CPD) of 50 cysts per kg of soil. For small infestation foci where the CPD is 5 cysts per kg of soil, the method has a detection probability of only 22 percent.

The delimiting rate for associated fields is 8,000 cubic centimeters (cc)/acre (ac), approximately 20 pounds (lbs)/ac. According to the SAMPLE model, for an infestation with a CPD of 50 cysts/kg in a field, the model shows a detection probability of 98.55 percent at the delimiting survey rate. Associated fields are required to undergo two surveys at the delimiting rate, each following a host crop. At a CPD of 50 cysts/kg, the second sampling detection should remain high. To calculate the cumulative detection probabilities with repetitive sampling, the product of both non-detection probabilities are combined. The probability of no detection each year is 1 − 0.9855 = 0.0145. If this happens twice, the combined probability of no detection equals 0.01452 = 0.00021025. Detection after two crops surveyed by this method is 1 − 0.00021025 = 0.9998, or 99.98 percent. For small infestations of 5 cysts/kg (approximately 2 cysts per Start Printed Page 34539pound) of soil, however, repetitive sampling is even more important because the detection probability starts at 22 percent but increases with each host crop.

The infested field monitoring survey rate is 80,000 cc/ac, approximately 200 lbs/ac. Because of the small infestation foci in Idaho, a declining cyst population from the absence of host crops, and the application of eradication treatments, intensive sampling increases the chance of detection and the accuracy of population estimation. As a result, the intense monitoring survey rate of 80,000 cc/ac for infested fields is scientifically supported.

The infested field in-field bioassay rate is 20,000 cc/ac, approximately 50 lbs/ac. This rate is scientifically justified by the model where a small infestation with a CPD of 5 cysts/kg has a detection probability of 22 percent. As described for the delimiting survey method, the model shows that when the CPD increases, the detection probability also increases. Because the in-field bioassay reintroduces host crops and requires soil surveys following each of three host crops, the incipient population increases; therefore, detection probability also significantly increases.

Soil samples are collected at the field surface; however, potato harvest machinery and annual tillage practices effectively mix the top layer of the soil such that soil samples represent at least the top 30 centimeters of the soil profile. PCN program sampling rates are higher than those used by many other countries where PCN infestations are widespread and have been present for decades. Lower sampling rates are generally used for managing high infestations and reducing economic impacts of the pest, not for eradicating nor limiting spread of the pest.

Infested Field Confirmatory Policy

To evaluate a field for PCN under the confirmatory protocol, a soil sample is required. Sanitary requirements for entering a field (boots, washing of tools), soil bagging and labeling, and vehicle disinfection are longstanding and widely observed practices used by APHIS to prevent the spread of plant pests from infected fields.

The protocol for determining infested field regulation for PCN is based on our knowledge about the biology and epidemiology of PCN. Specimens from a soil sample must be definitively identified and confirmed by an APHIS-approved laboratory using morphological and molecular DNA-based methods. Molecular methods provide an additional, confirmatory step along with morphological methods.

Details of APHIS' use of DNA and morphological/morphometric identification of PCN are described in a 2007 scientific article,[13] which is provided via a link in the confirmatory protocol. In the 1990s, nematologists began using DNA technology extensively for identification purposes, while morphological identification of nematodes has been widely in practice for decades. The technical minimum threshold for declaring a field infested/positive for PCN is met by detecting a minimum of two cysts from two samples that were identified as PCN by morphological/morphometric analysis, and at least one of the cysts was viable and confirmed as PCN by molecular (DNA) analysis. It is not necessary for the two samples to come from the same survey event.

Regulating Associated Fields

The protocol for determining associated field regulation is modeled in part after the USDA Golden Nematode Program and its criteria for determining “exposed land” as described in the USDA Golden Nematode Manual (2006 version).[14] Unlike the Golden Nematode Program approach of regulating large blocks of land or entire counties, the PCN Program adopted a more conservative field-by-field regulatory approach in which only confirmed infested fields and those at high risk for infestation are regulated. Associated fields are identified through the process of researching an infested field's history, going back 10 years, to identify other fields that may have been exposed to infested field soil.

Infested Field Deregulation Protocol (if Remaining in Host Crop Production)

Fields that APHIS has determined to be infested with PCN are eligible for release under a deregulation protocol if the field is used for host crop production. The infested field deregulation protocol employs strategies that have been used for decades to control nematodes on potatoes and other crops.

Fixed Grid Pattern Field Sampling

In the Infested Field Deregulation Protocol, APHIS conducts an initial full field survey in a fixed grid pattern at an 80,000 cc of soil per acre sampling rate. The sampling results (number of cysts per sample) are used to map the relative distribution and population of cysts in the field, and infestation foci are located. The fixed grid survey is a standard industry practice for monitoring several types of field activities, including mapping infestations and monitoring pest eradication treatments. For instance, one study APHIS drew upon in developing the protocols describes a method for PCN soil sampling by which a field is divided into 20 x 20 meter grid squares, then soil samples are collected from each grid. The samples are processed to separate cysts from the soil, and the number of cysts per grid is determined by counting. The results of the cyst counts are plotted to produce a map of the infestation across the field.[15] Identifying infestation foci informs soil treatment decisions and cost-effective monitoring of treatment efficacy over time. This method is the basis for the PCN program's mapping surveys and subsequent grid monitoring surveys.

The PCN sampling method for infested fields is based on a 2 x 2 grid pattern method (subsamples are collected 2 paces apart, every 2 paces) modeled in part after a grid survey method described in GN program manuals from 1992 and 2006. The 2006 manual describes the steps for such a survey, beginning with measuring the dimensions of the field, dividing the field into a grid, and sampling the soil following the grid pattern. If nematodes are located in a sample, the grid makes it possible to trace that sample back to a location in the field.[16]

After sampling results are determined, a field may undergo a series of optional, PCN program-sponsored eradication treatments, which are monitored according to initial grid survey results. These treatments are conducted at the discretion of the grower. Eradication treatments have included Telone® II fumigation and the trap crop litchi tomato. Telone® and Telone® II have been widely employed as a nematicide for control of all major species of nematodes throughout the United States, as has litchi tomato as a trap crop Start Printed Page 34540in other countries. Trap crops, which have been used for decades to control nematodes, can be effective in reducing yield loss in potatoes and other crops when used as part of a crop rotation, or in conjunction with the use of nematicides.[17]

Host crops may be grown consecutively or in a crop rotation. A field is eligible for full deregulation if no viable cysts are detected after each of three host crops are harvested.[18] The scientific rationale for requiring three crops is to allow multiplication and detection of any low-level PCN populations prior to release.

Viability Testing, Staining, and Bioassays

In the Infested Field Deregulation Protocol, initial cyst viability is assessed using a live/dead staining assay. The staining assay to determine viability is a standard procedure in nematology as it allows for clearer visual identification of the organism. To evaluate the efficacy of a treatment for cyst nematode control, determining if a nematode is dead or alive is important. The lack of movement of a nematode does not signify death in species like Heterodera spp. (cyst nematodes).[19] Since the egg is protected in a resistant structure, living (viable) and dead (nonviable) eggs cannot be distinguished by direct observation. Various dyes and stains have been used to visualize and then ascertain viability of nematode eggs.

To become deregulated, a field must complete a series of tests to demonstrate that the infestation has been fully eradicated. In classical nematology, the standard method to determine PCN viability is based on a staining assay, using Meldola's blue dye (MB) followed by microscopic visualization of MB‐treated nematodes. Nematode staining techniques are widely accepted by the majority of nematology laboratories and have been for decades.[20] One study presents a novel hatching bioassay technique developed for golden nematode, in which the authors illustrated the feasibility and advantages of a hatching bioassay system using staining and fluorescence microscopy. Another study [21] published in 1996 discusses the results of PCN infectivity assays using staining techniques similar to those we prescribe in the deregulation protocol. We also note that the 1968 USDA Golden Nematode Program Manual includes viability testing to monitor efficacy of chemical treatments.

As part of the infested field protocol, we also assess cyst viability using a greenhouse bioassay method (equivalent to three consecutive susceptible potato crops) or an in-field bioassay method (three consecutive crops grown in infestation foci or over the entire field). Greenhouse and field bioassays are used throughout the world to evaluate pest viability and other biological characteristics.[22]

Associated Field Deregulation Protocol (if Remaining in Host Crop Production)

The primary determination for a field to become regulated as an associated field is exposure of that field to soil or other regulated articles from an infested field. Pressure washing sanitation requirements, explained above, are implemented for all equipment in contact with field soil. These requirements are necessary to mitigate the potential spread of PCN from associated fields that are considered high risk for PCN infestation. Other regulatory requirements are implemented for movement of commodities and articles from the field that cannot be sanitized. For PCN, a full‐field delimiting survey at a sampling rate of 8,000 cc of soil per acre (equivalent to approximately 20 pounds of soil per acre) is used to determine its presence in associated fields. A series of two negative delimiting surveys, each following harvest of two host crops grown on the field, is required to deregulate an associated field. The current deregulation protocol was adopted by APHIS in 2012 at the request of cooperators and stakeholders that were impacted, including the Idaho State Department of Agriculture, Idaho Potato Commission, and owners and operators of infested and associated fields.

Delimiting surveys are a common practice that have been included in APHIS emergency response manuals and used by several APHIS, State, and international programs. For example, a Japanese Beetle Harmonization plan, adopted by the National Plant Board in 1998, uses the same concept as the PCN deregulation protocol of conducting detection surveys followed by a more robust delimiting survey. This Japanese beetle harmonization plan was implemented by the Idaho State Department of Agriculture in Boise, Idaho in 2013 after detection of the beetle in 2012.[23]

Deregulation Protocol for Agricultural Land No Longer in Host Crop Production and Non-Agricultural Land

A deregulation option exists for regulated fields where agriculture still occurs but where all host crop production was prohibited or has ceased for a minimum of 30 years. This could include infested or associated status fields. During the 30-year time period, the fields may have been used for various purposes, including but not limited to hobby farms, fallow fields, forage crops, grain fields, nurseries, or pasture. PCN can remain viable for approximately 30 years in the absence of a host crop.[24]

To become deregulated, fields no longer in host crop production must complete a two-step process. Records must be made available to APHIS to demonstrate that the land has been out of host crop production for the last 30 years. APHIS then surveys the entire field at a rate of 8,000 cc soil per acre (equivalent to approximately 20 pounds of soil per acre). This dual approach establishes a 30-year period in which the field is out of host production, making it much less likely that PCN is present, and in the present establishes whether any viable PCN remains.

A deregulation option also exists for regulated fields that have been converted to non-agricultural uses. This could include infested or associated status fields. Examples of non-agricultural uses include such things as highways and other paved roads and commercial, industrial or residential development.

To become deregulated, fields converted to non-agricultural uses must have records available to determine the land has been out of agricultural use for Start Printed Page 34541at least the last 20 years and will not return to production, or construction for non-agricultural proposes has rendered the land non-tillable and is not likely to return to agricultural production. The risk of PCN spread and establishment from these non-agricultural fields is lower than those remaining in non-PCN host agricultural production, resulting in the lower number of years required for release. In the APHIS Karnal Bunt Program, which has been in place since 1996, a similar provision in the regulations [25] has been used successfully to lower or eliminate the risk of Karnal Bunt if the land cannot be farmed.

In order to give the public an opportunity to consider the science on which we have established the field protocols and the sources we have used to develop them, we are reopening the comment period on Docket No. APHIS-2018-0041 for an additional 30 days. This action will allow interested persons additional time to prepare and submit comments.

Start Signature

Done in Washington, DC, this 21st day of May 2020.

Michael Watson,

Acting Administrator, Animal and Plant Health Inspection Service.

End Signature End Supplemental Information


1.  To view the proposed rule, supporting documents, and the comments we received, go to​#!docketDetail;​D=​APHIS-2018-0041.

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2.  Memorandum Decision and Order, Mickelsen Farms, LLC, et al. v. APHIS, et al., March 20, 2018.​fdsys/​pkg/​USCOURTS-idd-1_​15-cv-00143/​pdf/​USCOURTS-idd-1_​15-cv-00143-2.pdf.

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3.  Docket No. APHIS-2006-0143; 72 FR (51975-51988), September 12, 2007.

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4.  To view the manual on, see footnote 1.

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5.  See footnote 1 for a link to the protocols.

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6.  Banks, N.C., et. al. Dispersal of Potato Cyst Nematodes Measured Using Historical and Spatial Statistical Analyses. Phytopathology, Vol. 102, No. 6, 2012.

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8.  Brodie, B.B., Probability of Globodera rostochiensis Spread on Equipment and Potato Tubers. Journal of Nematology 25(2):291-296. 1993.

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9.  Brodie, B.B., and M.L. Brucato. Relation of Cyst Age and Egg Density to Establishment of Globodera Rostochiensis populations. Journal of Nematology 21:4 October 1989.

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10.  Stienstra, W.C., and D.H. McDonald. The Soybean Cyst Nematode. Minnesota Extension Service AG-FO-3935 1990.

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11.  Golden Nematode Program Manual (2006): 2-8-18. Similar steam and pressure cleaning requirements are included in earlier versions of the manual published in 1968 and 1992. All versions are available via the link to in footnote 1 of this document.

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12.  Additional descriptions of these sampling methods are: (1) Been, T.H. and Schomaker, C.H. 1998. Sampling methods for fields with patchy infestations of the potato cyst nematode (Globodera spp.): A simulation model to develop and evaluate sampling methods. In Quantitative studies on the management of potato cyst nematodes (Globodera spp.) in the Netherlands. p. 319; and (2) Been, T.H. and Schomaker, C.H. 2000. Development and evaluation of sampling methods for fields with infestation foci of potato cyst nematodes (Globodera rostochiensis and G. pallida). Phytopathology 90:647-656.

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13.  Skantar, et al., Morphological and Molecular Identification of Globodera pallida Associated with Potato in Idaho. Journal of Nematology, 2007 Jun; 39(2): 133-144.​pmc/​articles/​PMC2586493/​. In addition, a diagnostic protocol for Globodera rostochiensis and Globodera pallida (PM 7/40 (4)) was approved as an European Plant Protection Organization Standard in 2003 and last revised in 2017:​doi/​full/​10.1111/​epp.12391.

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14.  To view the manual on, see footnote 1.

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15.  See Evans. K., et al., Mapping Infestations of Potato Cyst Nematodes and the Potential for Spatially Varying Applications of Nematicides. Precision Agriculture 4 (2003) 149-162.

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16.  Golden Nematode Program Manual (2006): 2-3-7. To view the manual on, see footnote 1.

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17.  See Sparkes, Jessica, Potential trap crops for the control of Potato Cyst Nematode (PCN). ADAS UK Ltd. 2013:​sites/​default/​files/​publication_​upload/​PCN%20trap%20crops%20review_​for%20publication.pdf.

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18.  See Greco N., et al., The Effect of Globodera Pallida and G. Rostochiensis On Potato Yield. Nematologica 28.4: January 1982:​view/​journals/​nema/​28/​4/​article-p379_​2.xml.

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19.  Shepherd, A.M. 1962. New blue R, a stain that differentiates between living and dead nematodes. Nematologica 8: 201-208.

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20.  Perry, R. and Feil, J., Observations on a Novel Hatching Bioassay for Globodera Rostochiensis Using Fluorescence Microscopy. Revue Nématologie 9 (31): 280-282 (1986).

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21.  Zanna, Muhammad, Diapause in the nematode Globodera pallida. European Journal of Plant Pathology 100: 413-423, 1994.

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22.  See McKenzie, M.M. and S.J. Turner, Assessing reproduction of potato cyst nematodes (Globodera rostochiensis and G. pallida) on potato cultivars for National Listing. EPPO Bulletin 17:3: September 1987.​doi/​abs/​10.1111/​j.1365-2338.1987.tb00048.x.

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24.  Turner, Susan. Population decline of potato cyst nematodes (Globodera rostochiensis, G. pallida) in field soils in Northern Ireland. Annals of Applied Biology, October 1996:​doi/​abs/​10.1111/​j.1744-7348.1996.tb05754.x.

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25.  See 7 CFR 301.89-3(f)(1).

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[FR Doc. 2020-11792 Filed 6-4-20; 8:45 am]