Grape Commodity-Based Survey Guidelines

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Grape Commodity-based Survey Guidelines

11 August 2008 Last Revision: August 2010 Melinda Sullivan and Edward Jones USDA APHIS Plant Protection and Quarantine Center for Plant Health Science and Technology

Table of Contents

Chapter 1: Introduction…………………………………………………………………………………………………………………….…5

Purpose of Document 5

Location of Surveys 7 Time Frame 7

Organisms to be Surveyed 7

Chapter 2: Survey Design & Sampling Methodology .………………………………………………………………..…..9

Introduction 9

Summary of Action Steps 9

Objective of Survey 10

Population to be Sampled 10

Data to be Collected 10

Degree of Precision Re- 11 quired The Frame 12

Selection of sampling plan 13 and sample selection Methods and Units of Meas- 17 ure

Pre-test 19 The Organization of Field 19 Work

Summary and Analysis of 20 Data Gaining Information for Fu- 20 ture Surveys

Chapter 3: Summary of Survey Strategies………………………………………………………………………………….21

Visual Survey 21

Trapping 28

Chapter 4: Pest Tables…………………………………………………………………………………………………………..29

Pests by affected plant part 29

CAPS– approved survey 30 methods

Chapter 5: Detailed Survey Tables………………………………………………………………………………………………31 Adoxophyes orana 31 Autographa gamma 33 Copitarsia spp. 34

Diabrotica speciosa 35 Epiphyas postvittana 36 Eupoecilia ambiguella 37 Heteronychus arator 38 Lobesia botrana 39 Planococcus minor 40 Spodoptera littoralis 41 Spodoptera litura 42 Thaumatotibia leucotreta 43 Candidatus Phytoplasma aus- 44 traliense Phellinus noxius 45

Chapter 6: Identification Tables…………………………………………………………………………………………………46

Adoxophyes orana 46 Autographa gamma 48

Copitarsia spp. 51 Diabrotica speciosa 54 Epiphyas postvittana 56 Eupoecilia ambiguella 58 Heteronychus arator 60 Lobesia botrana 62 Planococcus minor 64 Spodoptera littoralis 65 Spodoptera litura 67 Thaumatotibia leucotreta 69 Candidatus Phytoplasma aus- 71 traliense Phellinus noxius 72

Appendix A…………………………………...…………………………………………………………………………………………………..74

Appendix B…………………………………………………………………………………………………………………………..81

Appendix C………………………………………………………………………………………………………………………………………. 86

Draft Log: The state of science is advancing faster than most documents in print can be updated. As such, this document is entitled “perpetual draft” with the intention of making updates as they are appropriate. Updates might include the inclusion of new high risk pests that are identified, inclusion of new survey methodology for existing pests, or removal of current pests that no longer pose a high risk or have become well established in the United States. In order to keep track of changes in the document, please find the draft log listed below. The date of the current draft is listed on the cover page.

Original Submission: August 2008 Revised Submission: October 2008

July 2010: Added Diabrotica speciosa and Eupoecilia ambiguella. Added Appendix M information and removed outdated information. Updated survey chapter. Fixed several typographic errors.

August 2010: Updated hyperlinks.

Chapter 1: Introduction

Purpose of Document: Welcome to the Grape Commodity-based Survey Guideline. This document is intended to be a tool to assist you as you develop pest detection survey plans in your respective states for exotic pests of grape. A detection survey determines the presence or absence of a pest but does not delimit a pest or establish the prevalence of a pest. This is a companion document to the Grape Commodity-based Survey Reference, available at the CAPS website (Figure 1.1). The Grape Commodity-based Sur- vey Reference is a collection of detailed datasheets on 26 exotic pests, 6 pests of restricted distribution in the United States with regulatory significance, en- demic pests easily confused with exotic pests, and potential vectors of exotic pests. These datasheets contain information on the biology, host range, survey strategies, and identification of these pests.

The Grape Commodity-based Survey Guideline is the result of a concerted effort to help states focus resources on survey efforts and identification of a smaller group of target pests. This guide contains little information about biology. We must acknowledge that there is no silver bullet survey that would be wholly applicable to each state. Environment, personnel, budgets, and resources vary from state to state. However, as state participants in the Coop- Figure 1.1 Cover Page of the erative Agricultural Pest Survey (CAPS), you can take steps to increase the uni- Grape Commodity –based Survey Reference. This document con- formity and usability of data across political, geographic, and climatic regions, tains pest datasheets on the most while maintaining flexibility for appropriateness within individual regions. This threatening exotic pests of grape manual is not intended to be a field guide to identify exotic pests in the field including information on biology, and distinguish the exotic pest from commonly occurring pests. The purpose of survey, and identification. The document is available for this manual is to provide a framework to aid cooperators in collecting the best download from the CAPS website. samples to send to a qualified taxonomist or diagnostician for pest identification. Considerable diagnostic or taxonomic expertise may already exist in your state. The survey methods described in this document combine survey strategies for exotic pests, including arthropods and plant pathogens. It is important to note that these broad categories have unique biological features that dictate current methods of survey. However, each category is grouped according to an appropriate sampling method within the context of this manual.

This Commodity-based Survey Guideline is intended to be implemented over several years with the initial field survey year beginning in FY 09. Portions of the recommendations may need clarification or adjust- ment as funding levels change, new threats are identified, or detection technologies improve. The transition to commodity based survey has just be- gun, and as such, end user feedback will be impera- tive to the creation of a useful end-product for corn and other commodities of large economic impor- Figure 1.2 Figure of the United States showing grape acreage by tance. National survey methodologies as estab- county in 2007. This image was generated using data from the Na- lished will take precedence over the methods de- tional Agricultural Statistics Service by Daniel Borchert (USDA-APHIS- scribed in this manual. Methods listed in Appendix M PPQ-CPHST). of the National Survey Guidelines will also take precedence over the methods described.

Chapter 1: Introduction

Table 1.2. Pests targeted in the CAPS Grape Commodity-based Location of Surveys: Note that the lo- Survey Guideline. cality scope of the Survey Guideline is limited to the contiguous United States. The acreage by Scientific Name Common Name Type of Pest county is shown in Figure 1.2. The total bearing acreage and the value for the top 5 producing states for grape in this manual is shown in Fig- Adoxophyes orana Summer fruit tortrix Arthropod-Moth (minor pest) ures 1.3, 1.4, respectively. Total bearing acreage by type of grape and value of production is shown in Figures 1.5, 1.6, respectively for Cali- Autographa gamma Silver-Y moth Arthropod-Moth fornia, which accounts for greater than 80% of (major pest) the total acreage and value for the United States.

Copitarsia spp. Owlet moths Arthropod-Moth (major pest) Time Frame: The Survey Guideline is intended to be carried out as a multi-year survey. Diabrotica speciosa Cucurbit beetle Arthropod– Beetle State and federal priorities, resources, and (major pest) funding, however, may influence whether the survey can be carried out for multiple years. Future versions of this manual may call for on- Epiphyas postvittana Light brown apple Arthropod-Moth going surveys following the same or a slightly moth (major pest) modified protocol. The multi-year time frame has advantages because states will have more Eupoecilia am- European grape berry Arthropod-Moth opportunity to collect data over a larger time- biguella moth (major pest) scale. Negative data collected over several years using a statistically based protocol can be influential in scientific, political, and trade Heteronychus arator African black beetle Arthropod– Beetle arenas. (major pest)

Lobesia botrana European grapevine Arthropod-Moth Organisms to be Surveyed: The moth (major pest) scope of surveyed organisms within the Survey Guideline is limited to a sub-group of pests from Planococcus minor Passionvine mealybug, Arthropod- the Grape Commodity-based Survey Refer- Pacific mealybug mealybug (major ence from the FY 09’ and 10 Analytical Hierar- pest) chy Process (AHP) Prioritized Pest List and one emergency pest (light brown apple moth). This Spodoptera littoralis Egyptian cotton leaf- Arthropod-Moth sub-group includes 12 arthropods and 2 plant worm (major pest) pathogens (a phytoplasma, and a fungus). The scientific name and common names of these Spodoptera litura Rice cutworm Arthropod-Moth pests are shown in Table 1.2. Photos of each major pest) pest are given in Figure 1.7. Many of the pests targeted in this survey can be Thaumatotibia leuco- False coding moth Arthropod-Moth detected visually or by collecting samples of treta (minor pest) plant tissues. As a result, 1-2 trips for each survey should be adequate. For most of the arthro- Candidatus Phyto- Australian grapevine Phytoplasma pods, pheromone lures are available, and use plasma australiense yellows (major pest) of these lures with traps will require a minimum of two trips per site. Field personnel are encour- Phellinus noxius Brown root rot Fungus (major aged to inspect for foliar pests during each trip. pest)

Note: With the exception of Epiphyas postvittana and Lobesia botrana, no other pests in this guideline have been detected in the continental United States. Both moths have been detected in California and eradication efforts are ongoing.

6 Chapter 1: Introduction

1,000,000 3,500,000 900,000 3,000,000 800,000 700,000 2,500,000 600,000 2,000,000 500,000 400,000 1,500,000

Bearing Acreage Bearing 300,000

Value (1,000 dollars) 1,000,000 200,000 100,000 500,000 - 0 Total CA WA NY OR PA Total CA WA NY OR PA U.S. U.S. State State

Figure 1.3 Grape bearing acreage for the United States and the top five produc- Figure 1.4 Utilized production value acreage for the United States and the top tion states in 2006. Data from the National Agricultural Statistics Service. five production states in 2006. Data from the National Agricultural Statistics Service.

500,000 2,000,000 1,800,000 400,000 1,600,000 1,400,000 300,000 1,200,000 1,000,000 200,000 800,000 Bearing Acreage 100,000 600,000 400,000 Value (1,000 dollars) (1,000 Value 0 200,000 Wine Table Raisin 0 Wine Table Raisin Type Type

Figure 1.5 Grape bearing acreage by type of grape for California in 2006. Figure 1.6 Utilized production value acreage by type of grape for California in Data from the National Agricultural Statistics Service. 2006. Data from the National Agricultural Statistics Service.

Chapter 1: Introduction A B D

C G E F

H I J

K M

Figure 1.7 Grape pests to be surveyed for as part of Grape Commodity-based Survey Guidelines. A. Adoxophyes orana, B. Autographa gamma, C. Candidatus Phytoplasma australiense, D. Copitarsia spp., E. Diabrotica speciosa, F. Epiphyas postvittana, G. Eupoecilia ambiguella, H. Heteronychus arator, I. Lobesia botrana, J. Phellinus noxius, K. Planococcus minor, L. Spodoptera littoralis, M. Spodoptera litura, and N. Thaumatotibia leucotreta.

Chapter 2: Survey Design & Sampling Methodology

Introduction: The purpose of this grape survey guideline is to provide guidance and to recommend the number of samples that will allow you to state with confidence that any one of these pests is below a specified incidence in your state. The methodology described was chosen to ensure a degree of statistical confidence without the requirement of a large, non-economically feasible sample size. The guideline outlines the steps involved to conduct a survey and gives you several options or alternatives to reach a desired goal. The steps described in each section can be accomplished through a number of methods. Keep in mind that these are only possible options. Each state will have to evaluate the options to find the best system to fit their limited resources to meet the objective. Often times there are many paths to get to the same endpoint. We recognize that many states utilize GIS and complex databases and computer systems in their survey activities. Please feel free to use the methodology that is most convenient to use in your state. In some cases, these may be a good fit for Visual Sampling Plan frame and sample development. In other cases, the Na- tional Statistical Service or Farm Service Agency frames, the state may need to contact agencies well in advance to determine if data are available and what steps need to be completed to obtain the data. This procedure will outline the steps to develop a survey procedure and will cover:

1. the objective of the survey, 2. the population to be sampled, 3. data to be collected, 4. degree of precision required, 5. the frame, 6. selection of sampling plan, 7. sample selection, 8. methods and units of measure, 9. the pre-test, 10. the organization of field work, 11. summary and analysis of data, and 12. documenting information gained for future surveys.

Summary of Action Steps Required to Conduct Survey: 1. Determine the distribution of fields/vineyards (herein referred to as vineyards) in your state. The first questions to ask yourself are: 1. How much grape production is in your state? and 2. How is it distributed across the state by county?

2. Determine how many samples you need to take to survey for exotic pests. First you will need to determine the confidence level (90, 95, or 99%) and detection level (1%) that you desire (Table 2). In most cases, a 95% confidence level with a 1% level of detection will be a good place to start.

*If there is a need for a higher confidence level or a higher detection level, the sample size will in crease dramatically, and the survey may become cost prohibitive.

*Keep in mind that you may need to increase the sample size for refusal rate, inspection effectiveness, and deadwood described later.

3. Identify the counties where samples should be placed and number of samples per county, making sure to distribute samples evenly within each selected county.

*If you have international ports in your state, international trade zones or inter-borders with cropping areas adjacent, you may want to allocate more samples in these areas/counties. The prevailing winds may also influence where you want to allocate samples.

Chapter 2: Survey Design & Sampling Methodology

4. Determine the locations (vineyards within a given county) that will be sampled and arrange for permission from growers to conduct visual surveys and set up traps. What is the best means available in your state to get a map of growing areas or a list of growers and conversely the vineyards within each grape growing county within the state? How each state chooses to do this may vary but the end goal should be detecting grape pests at a specific level of infestation with a specific confidence level.

*Contacting NASS (they will not share their list of growers) for possible land use samples, contacting grower associates, or mapping grape or vineyard areas of your state may aid in determining the locations to be sampled.

*In addition, a process must be followed to select the vineyards for sampling. The process needs to be well-defined and the decision of which field to sample should not be left in the hands of the individual conducting the survey. Following the process should guide them to the sample field.

5. Once you have the list and have selected vineyards, you must obtain permission from the growers to conduct a survey in the field.

Steps to Develop Survey Procedure: 1. Objective of Survey: The survey objective is to establish the presence or lack of presence of selected grape pests in the states surveyed. This is where you determine which pests to be targeted with the survey. To ac- complish this objective, grape target pests will be detected in and around the vicinity of grape vineyards. This survey is intended for states with at least 1000 tons of production.

2. Population to be Sampled: The population to be sampled is grape fields/vineyards (herein referred to as vineyards) and the areas immediately adjacent to vineyards within a grape producing state. You may also want to divide the regions or counties into different groups based cultivation practices, type of grapes grown, and risk of introduction based on known pest pathways. The key factors in population sample identification are to:

Determine the distribution of fields/vineyards (herein referred to as vineyards) in your state. The first questions to ask yourself are: 1. How much grape production is in your state?, 2. How is it distributed across the state by county?, and 3. Are there high risk areas?

Terminology: We are sampling vineyards. Vineyards will have sample plots in them. The vineyard is the sample unit. The plots in the vineyards or adjacent to the vineyard are subsamples associated with the sample unit. *A sample vineyard is a farm containing one or more sample vineyards or vineyard blocks. *A sample county is a county containing one or more sample vineyards. *A population refers to the population of vineyards within a state. If we sample from the population of vineyards within a state, we can extrapolate back to the population from which the sample was selected. If we find a pest within our samples, we can extrapolate to the population of pests within our population of grape vineyards.

3. Data to be Collected: We must decide what data is to be collected. The data to be collected will be driven by the survey objective target pests and the population distribution of vineyards.

Chapter 2: Survey Design & Sampling Methodology Key data may include: What pest(s) is the survey aimed to find, survey date, survey time, survey type (visual, trapping, sweep net, black light), trap type, attractant/lure used, trap number/identifier, latitude, longitude, sample submission date, sample submission time, symptoms observed (if any), plant part affected, level of infestation, sample identification number, plant part collected, stage of organism found (egg, larva, pupa, nymph, adult, all stages, unknown, condition (live, dead), diagnostic elements (name of diagnostic laboratory, specialist), and diagnostic methodology (morphological, ELISA). Will a plant tissue sample be collected? Will a soil sample be collected.

We recommended that the data collected concerning each sample is reasonable and that data only be collected that are necessary to track a sample and make sound decisions.

Each PPQ regional office will work with the states to develop and provide a template to the states on what data are to be collected at the time of a survey with key sample elements.

4. Degree of Precision Required: The level of infestation to be detected establishes the required precision of the survey. For grapes, we are recommending as a standard that you detect a 1% or smaller infestation. In- dividual states may find this level of precision difficult to attain due to time and cost issues. These issues may result in states deviating from the standard. Table 2 .1 shows the sample size required to achieve a certain degree of precision. The degree of precision is one of the primary factors in determining the sample size.

In cases where resource limitations limit sample size the degree of precision becomes the result of a doable sample size rather than the driver of sample size. Again Table 2 .1 is provided as a guide to the relationship between sample size and infestation detection level.

5. The Frame: Frame development is determining the locations (vineyards within a given county) that will be potential sample unite for visual surveys and set up traps. What is the best means available in your state to get a map of growing areas or a list of growers and conversely the vineyards within each grape-growing county within the state? How each state chooses to do this may vary but the end goal should be detecting grape pests at a specific level of infestation with a specific confidence level. But first here is some information you need to know about frame development.

A frame is a device used to represent the population. The frame is made up of frame units, which represent members of the population. The frame units must be defined so that there is a clear distinction between frame units. Each frame unit must be mutually exclusive from any other unit in the frame. No part of a frame unit should be considered part or confused with another frame unit. The sample is selected from the frame. The frame units selected for the sample become sample units.

In this case, grape pest detection, the vineyards or vineyard blocks become the frame unit. The vineyard or vineyard block is an area of continuous cultivation much the same as a field would be defined. If a single grape growing operation often referred to as a vineyard has more than one vineyard block (field) than each vineyard block would be considered as a frame unit.

In a given state the fields or field blocks must be listed, mapped, or otherwise organized into a frame so that we can apply a selection process to select our sample.

There are no perfect frames. The frame will be incomplete, contain duplication or both. Some frames may also be very difficult to work with. List frames tend to be incomplete or have duplication or both. Map frames can be more difficult to work with; however, today’s more computerized digital images can make this task much easier.

For grape, there are at least five different frame alternatives listed below. Each state will determine which frame will work best for their situation.

Chapter 2: Survey Design & Sampling Methodology A. National Agricultural Statistical Service (NASS) frame: In states where NASS does a vineyard survey they may have a listing if growers that use for sampling. You may be able to work with them as a state cooperator get a listing of the growers or work with them too select a sample of vineyards. It will also help find growers who have been prescreened and will be more likely to cooperate with the survey.

NASS also has an area frame ; however, it is most likely not well suited for a vineyard sample. This frame will only be useful for states with large grape acreages, for example California.

The NASS area frame is based on land use. NASS takes all land in a state and classifies the land tracts based on land use (e.g. urban, intense agriculture (ag), moderate ag, light ag, forest, etc.).

NASS then further classifies these tracts into segments. This allows NASS to set up an effective area

frame based on land use to select a sample. NASS samples its area frame each year in June. Dur-

ing this June survey, NASS identifies fields in the sampled segments planted to grapes. Aerial photography of fields and county maps of where segments are located within a county are available within the NASS office. They also can identify farmers who would be willing to allow pest detection sampling. NASS also can select a sample of vineyards to be used in our pest detection survey. This provides a possible means to facilitate selection of a high quality sample for pest detection. With proper planning and coordination, these samples should be able to be made available to state cooperators.

If a state would like to try to use one of the NASS frames, we suggest that you contact Edward (Ned) Jones with CPHST ([email protected]) to help facilitate the process.

B. Farm Service Agency (FSA): This agency has aerial photography of farm land. Each year farmers come into the FSA Offices and identify the fields and crops that they intend to plant. State cooperators may be able to work with them to develop a frame of vineyards. This is not a proven concept but may have potential. We believe that this information may not be well-organized and has not been used for this purpose previously. However, this may be a useful tool to obtain a list of grape vineyards within a county or state. The FSA information could be used to identify grape growers at the county level. The growers could then be contacted and permissions obtained to enter their fields to conduct the pest detection survey.

C. County Acreage Estimates: NASS has county acreage estimates for grape in states where production is significant enough to warrant such information. This information could be helpful to allocate samples to counties.

D. Visual Sampling Plan (VSP): VSP is a free program available via the internet (http://dqo.pnl.gov/) that can be used to develop a map-based sampling frame. It was developed for the Department of Energy, Environmental Protection Agency and the Department of Defense. It is also now sponsored by the Department of Homeland Security and The Center for Disease Control. This program will provide information on the sample size required for a specific level of detection and where to locate the samples within a geographic area. VSP requires that you input digital maps. If you do not have access to digital maps of your state or counties within your state, some digital maps are available from the following Natural Resource and Conservation Service website (http://datagateway.nrcs.usda.gov/).

Note: to access this website you must login using a USDA e-Authentification (see http://www.eauth.egov.usda.gov/index.html for details on how to get an e-Authentification).

The program will also allow you to incorporate and identify roads, water ways, and other high risk areas. The program may not be user-friendly without a statistical sampling background and training in VSP; however, once a frame is set up in VSP, it allows a great deal of flexibility in developing a

Chapter 2: Survey Design & Sampling Methodology sampling plan. An example of the visual sampling output is shown in Appendix B. Note: the VSP example refers to room because it is set up for room sampling; however, it will do an excellent job with state or county maps. If a state would like to try to use this program, we suggest that you contact Edward (Ned) Jones ([email protected]) with CPHST to help facilitate the process.

E. Grower Associations and Wineries: Grape grower lists may be obtainable from various grower associations within a given state. The issues with this source and other similar sources of information include: completeness of the data, over-completeness (duplication) of the data, and the inclusion individuals or entities that are deceased or no longer in the grape business. Such problems are usually referred to as “deadwood.” States will need to increase their sample size to allow for these weaknesses in the frame. If acreage data is available on this list, it could be used to allocate the samples in a manner similar to that used to with the county estimate data.

6. Selection of sampling plan and sample selection: The sampling plan will be based on detection sampling. In detection sampling theory, the sample size is based on detecting a specified level of infestation (e.g. 1%), the effectiveness of the inspection method applied, and selecting a sample with a specific level of confidence (probability) that the specified level of infestation would be detected. A refusal is a sample unit whose owner denies permission to sample his/her vineyard. The refusal rate r is the proportion of the sample which become refusals (r=(sample refusals)/(total sample). By including re, the expected refusal rate, to the equation the sample is also adjusted for the expected refusal rate. The sample size can be estimated as follows: ner = ln(1-Pr(a>0)) (1- re) ln(1-(eP))

Where: ner = the detection sample size (number of frame units, i.e. vineyards or vineyard blocks) adjusted for inspection effectiveness and refusals,

P = the level of infestation to be detected,

e = the efficiency of the inspection process,

re = the expected refusal rate (expected=best guess), and

Pr(a>0) = the probability (confidence) the sample will detect a P infestation, given an “e” inspection efficiency,

ln(….) = the natural logarithm of the value in brackets (…..).

The detection sampling plan used will also be influenced by the frame selected. Each frame will require a slightly different sample selection plan, depending on conditions in the state, knowledge of the frame, and the quality of the frame. A. The NASS Frame: NASS locates the samples and could do some up front work to obtain permission for the CAPS samples. Utilizing the NASS frame will require coordination in advance with the state NASS office. Six months lead time before June would be a smart approach.

B. Farm Service Agency Data: Samples would be selected by probability proportional to size of the grape acres in the county.

First distribute the samples per county based on the number of FSA grapes planted per county (see NASS county estimates example in Appendix C and C below for a specific example of probability

Chapter 2: Survey Design & Sampling Methodology

proportional to size). At the county level, list the vineyard IDs and the acres planted to grapes. Sort the list based on planted acres (largest to smallest). Develop a cumulative summary of the acres to determine the field-by-field (or vineyard-by-vineyard) cumulative total acres (if possible) planted to grapes in the county. The cumulative total for the last field (vineyard) in the listing will be the total for the county. At the county, level divide the number of samples into total acres planted to develop a ‘county acres skip interval’.

Develop a random start by multiplying a random uniform number between 0 and 1 times the ‘county acres skip interval’. Use a table of random numbers from a reliable statistics book to generate the random number. DO NOT USE EXCEL as its random number generator is not reliable. The result will be a random start. This is the first sample point. It should correspond to the field (vineyard) with al least that many cumulative acres. This is the first sample unit in the county. Add the ‘county acres skip interval’ to the random start to obtain the cumulative acres for the second sample point. It should correspond to the field (vineyard) with al least that many cumulative acres. This is the second sample unit in the county. Continue adding the ‘county acres skip interval’ to identify each of the cumulative sample points and corresponding sample fields. To ID the selected field, find the first field in the cumulative listing whose cumulative acres planted exceeds the cumulative sample point. Repeat this field selection to ID all selected fields in the county.

Two months lead time before May would be a smart approach. In some counties with large operations more than one sample unit will be located within a vineyard operation. When this occurs the operation will be made up of several vineyard blocks, the field worker will need to number the blocks and use a Random number table to select the blocks (sample units) for sampling. A similar approach can also be used to select a single sample unit (block) from a large operation.

C. NASS County Estimates Use the NASS County Estimates to allocate the samples to the counties. An example of allocating the estimates by probability proportionate to size is provided in Appendix C for California grapes acreage in 2002. The steps are as follows:

The approach here is to divide the state’s total acreage by the total number of samples for the state. The result is an ‘acreage sample interval’.

*For the CA example, the total acres were 888, 253. Divide this number by the total sample size, 300 samples in this case. The result is an ‘acreage sample interval’ with 2960.84 acres per sample. 2. Develop an acreage cumulative total for each county estimate. *See column 4 in example. In this example, Fresno County has an acreage of 232,659 acres. Kern has an acreage of 96,510 acres. The cumulative acreage for these two counties is 232,659 + 96,510, which is equal to 329,169. Continue this for all counties. 3. Divide each county acreage cumulative total by the acreage sample interval (from step 1). The result is a cumulative sample by county. Round the samples to the nearest whole sample.

*For the Fresno County example, divide the 232,659acres by 2960.84 samples/acre from step number 1. For Kern County, divide the 96,510 acres by 2960.84 samples acre. The result is 78.58 for Fresno Co. and 111.17 for Kern Co. Round each number to nearest whole sample (given here in column 6), where Fresno Co. would have a cumulative sample of 79 and Amador Co. would have 111. 4. Back out each county’s sample size by subtracting the previous county’s cumulative sample from the county’s cumulative sample. This result will be the number of samples needed for that county. Con- tinue this process for all the counties.

Chapter 2: Survey Design & Sampling Methodology

Table 2.1 Sample size required to reach a desired level of precision.

Confidence Inspection Detec- Sample size Sample size Sample size Sample size (%) Effective- tion no refusals 20% refusals 30% refusals 50% refusals ness threshold (%) 95 100 1 299 374 427 598

99 100 1 459 574 656 918

90 100 1 230 288 329 460

95 50 1 598 748 854 1,196

99 50 1 919 1,149 1,313 1,838

90 50 1 460 575 657 920

95 40 1 748 935 1,069 1,496

99 40 1 1,149 1,436 1,641 2,298

90 40 1 575 719 821 1,150

95 25 1 1,197 1,496 1,710 2,394

99 25 1 1,840 2,300 2,629 3,680

90 25 1 920 1,150 1,314 1,840

Chapter 2: Survey Design & Sampling Methodology *For the Fresno and Kern Co. example, Fresno would have 79 samples and Kern Co. would have 32 samples (111-79=32). The cumulative sample for Madera., the next county on the list, is 140. So, the county sample for Butte Co. is 29 (140-111=29).

Step 4 takes you through allocating the samples to the counties. Next a process must be developed to select the fields for sampling within the county. VSP may be able to provide a solution. It can identify a set of GPS coordinates for sample points. This process will only be applicable in counties with high densities of vineyards. If the vineyards are not dense, locate the vineyards using a local source such as a county agent. Then sample some or all of the vineyards as required by the number of samples needed for that county.

If you do not have a process to follow which distributes the samples evenly within each county, step 5 starts a suggested process.

5. Identify the vineyard areas within each county. Select a set of either north-south roads or a set of east-west roads within the vineyard area. In eastern states, a landowner meets and bounds system of land ownership is used (not township section), which may make identifying the need roads in vineyard areas more difficult.

6. Layout a serpentine course along the roads.

* Example: For an east- west road system, start in the northeast corner and proceed east on the northern most road the point until it leaves the vineyard area. Then turn south to the next east- west road and proceed west back to the other side of the vineyard area. Use this process to develop a route through the vineyard area(s) of the county. You may need to skip areas that are not vineyard areas (towns, cities, forestland, etc.).

7. Determine the total vineyard distance along the serpentine route, excluding the non-vineyard areas.

8. The total distance from step 7 should be divided by the number of samples allocated to the county. This will result in a vineyard route sample skip interval.

9. The skip interval should be applied to the route to identify the sample start points. If desired, a random start can be applied to the skip to randomize the first start point.

10. With the sample start points identified in step 9, the individual conducting sample would proceed along the route to the first sample point. If a vineyard was present at that point, it would become the sample field. If not, proceed along the route to the first vineyard visible on the right. Let this first field to the right become the sample field. If no vineyards are on the right before the next sample start point, then at the next sample start point turn around and proceed back to the original sample start Point. Looking for vineyards on the right. Note: If no field is found, record ”no field found” for sample number XXX. If no field found is a frequent occurrence, you may want to re-evaluate your vineyard route.

11. The same process would be followed from each sample start point.

D. Visual Sampling Plan - a Map-Based Solution VSP can determine the appropriate sample size and locate the samples spatially based on a number of strategies. VSP will develop a list of GSP coordinates to identify sample points. A strategy must be developed to identify a vineyard from the sample point (example: proceed north to the first vineyard sighted).

Chapter 2: Survey Design & Sampling Methodology

E. Growers Association Lists Using the growers association lists, the samples could be allocated to growers across the state using the same strategy employed to allocate samples at the county level with the FSA data. After the sample growers are identified, it may be necessary to identify fields for growers with more than one field.

Sample size: The sample size will be based on the required detection level and the desired confidence level of the sample. The confidence of the sample is the probability that a sample of a specific size will detect an infestation at the required detection level or larger.

This sample size can be applied to the entire state acreage to infer that the level of infestation is less than 1% in the entire state with 95% confidence or it can be applied to a smaller area even down to an individual field. The area defined as the population from which the sample is taken is the area we can make a statement about the level of infestation (an inference). If no infestations are found, we are 95% confident that any infestation is less than 1% in the area sampled. Table 2.1 shows the required sample size for given confidence and detection levels and refusal rates at four levels of inspection effectiveness. As a general rule, assuming 100% inspection effectiveness is ambitious to say the least and not a reasonable or practical approach. Inspection effectiveness is rarely better than 90% and usually values from 20-80% are more realistic. Try to apply a realistic evaluation of inspection effectiveness.

7. Methods and Units of Measure: Follow the guidelines provided by the Light Brown Apple Moth (LBAM) Na- tional Survey Plan to determine how many samples and where to place traps for LBAM http:// www.aphis.usda.gov/plant_health/plant_pest_info/lba_moth/downloads/lbam-natlsurveyguidelines.pdf.

The methods that follow are for pests other than LBAM:

Where to locate sample field/vineyard plot? Once vineyards are selected for sampling from your state and permission obtained, we recommend that visual surveys be conducted based on a rows and paces system similar to those used for pest scouting. The rows and paces system will be used to identify the subsample or plot area(s) of each vineyard to be inspected. Two to four subsample aisles (aisle, the area between two rows) will be located in each vineyard. In each subsample aisle, lay out 1 or 4 subsample units or plots depending on the length of the row and available resources. The predetermined number of paces could be re- selected randomly for each subsample date; however, it you feel that you need to revisit the same area multiple times throughout the growing season, it is recommended that a stake with flagging be used to mark the area of the vineyard. The subsample units in each row will be laid out using the following steps:

1) The aisle between the 1st and 2nd rows should be selected and the aisle between last and next to last row of the vineyard.

2) The other one or two aisle(s) should be chosen based on a count of the number of remaining row aisle applying a random uniform distribution between 0 and 1. (Hint: Multiply the random number times the number of row aisles and round any fractional product aisle up to the next whole row aisle. Exam- ples 4.3 round to 5, 4.7 round to 5, etc.)

3) The subsample plot would be located by counting off the number of row aisles before you step off a predetermined number of paces into the vineyard. A random number table can be used to determine these numbers and to eliminate bias. The number of paces down the row aisle should be based on the size of the vineyard block being sampled. The pace count will start at the beginning of the row. If there are more rows or paces than there are in the field/vineyard, simply use the bounce back method. For paces, turn around and count paces in the other direction.

4) When the count is reached, put a chaining pin down at the end of the count.

Chapter 2: Survey Design & Sampling Methodology 5) From that pin measure, 10 feet further down the aisle using the tape measure. Insert a chaining pin at the 10’ mark. Lay the stadia rod down perpendicular to the rows at the 10’ pin.

6) The first vine or vine cluster in each row beyond the rod will mark the start of the subsample unit. Since the grape vines are intertwined in the row, identifying unique plants in the subsample unit will not be possible or practical. The subsample unit will begin in the center of where the 1st vine or vine cluster emerges from the ground. The subsample unit will end at the point where the 6th vine or vine cluster emerges from the ground. The subsample unit extends from an imaginary vertical plane perpendicular to the row through the center of the 1st plant to a plane through where the center of the 6th plant emergence from the ground. A separate layout should be used for the row on the left and the row on the right. The subsample unit’s length may differ from the row on the right of the aisle to the row on the left of the aisle. This will depend on the plant spacing in the row or whether plants are missing.

7) Place a stake with flagging ribbon by the 1st and 6th plant in each row so that the unit can be easily located on future visits. Be sure to collect the chaining pins to use to lay out the next subsample unit. Permis- sion should be obtained to use stakes to identify the subsample plot location and must be removed at the last inspection visit before harvest.

8) From the end of the first subsample unit, pace off 100 paces to lay out a second unit. At the end of the 100 paces, follow steps 4 through 6 to lay out the subsample unit.

9) If four subsample units are laid out per row, the third subsample unit should be laid out 100 paces beyond the second subsample unit by following steps 4 through 7 to lay out the subsample unit.

10) Lay out the last unit (third or forth depending on the number units per aisle) by walking to the end of the aisle, turning around (about face), placing a chaining pin at the end of the aisle, and following steps 4 through 7 to lay out the subsample unit. If any part of the last unit overlaps a previously laid out subsample unit, do not lay out the last subsample unit.

In each subsample unit, visually examine vines and foliage between the 1st and 6th vine in detail. Look for obvious plant disease symptoms (yellowing, streaking, damage, dead tissue) and signs of pests (frass, larvae, adults). Make sure to examine vines/stems, leaves (including the underside), flowers (if present), and fruit for symptoms and signs of pests. Plant samples should be collected when specific symptoms and signs (that are outlined for the specific exotic pests in the detailed survey tables in this document and the grape reference guide) are observed. The template provided by the regional offices should be used to record the proper data on each subsample collected. When follow up visits are made, revisit the same plot. Use the original rows and paces to find the plot. If the plot cannot be found, relocate the subsample using the original rows and paces.

How many subsample plots per field/vineyard and how to subsample within the field/vineyard? Two or four subsample aisles (aisle, the area between two rows) will be located in each vineyard. In each subsample aisle lay out 1 or 4 subsample units depending on the length of the row and available resources. Alternative Method: Another option is to step off the random number of rows previously determined to make a transect through the vineyard or farm on that row. Plants would be inspected for symptoms and signs at a 10 foot interval. This should be repeated a minimum of three times.

Regardless of sampling method used, you should collect something obvious even if it falls outside the recommended sampling unit/area (including areas adjacent to the vineyard). In addition, you need to ac- count for zeros. If nothing is observed, it must be documented.

Visual Survey: This type of survey involves the examination of the grape plant for diagnostic symptoms (Fig. 2.1). In addition, the surveyor should also look for physical evidence (signs) of the pest (Fig. 2.1). The

Chapter 2: Survey Design & Sampling Methodology surveyors should pay close attention to symptomatic plants first. These would be the plants that have a chlorosis (yellowing), feeding holes, or a generally unhealthy appearance. If no symptomatic plants are present, the surveyor should choose plants to examine based on convenience. While the surveyor will examine several plants within the site, only one data recording will be necessary for the site. The surveyor may make at least 2 trips to each survey site during the survey season (see trapping, below), and will thus have the opportunity to twice conduct a visual survey. In the context of the current survey, surveyors should take note of the general condition of the plants, and further examine stems, leaves, flowers, and fruit for the pests of concern. Subsample sites need not be the same on subsequent visits.

Trapping: This type of survey involves the use of a trap to catch arthropods of interest in a specific location. Often times, trap efficiency is increased through the use of some type of chemical or physical attractant (e.g. pheromone lures). In the context of the current survey, there are nine arthropods which can be surveyed via this method. The nine include Adoxophyes orana, Autographa gamma, Copitarsia spp., Epiphyas postvittana, Eupoecilia ambiguella, Lobesia botrana, Spodoptera littoralis, Spodoptera litura, and Thaumatotibia leucotreta. However, the commer- Figure 2.1. Top: Example of plant cial availability of the pheromone for Copitarsia spp. is unknown. Copitar- chlorosis (a symptom), Bottom: ex- sia spp., have traditionally been trapped via black light trapping for early ample of eggs and neonates (a detection surveys and is the CAPS-Approved Method for survey for this sign) pest.

Handle and store pheromone lures appropriately, or they will not be effective. Lures need to be kept in their sealed packages until ready for use and should be stored in the refrigerator or freezer as directed by the manufacturer. Use gloves to avoid cross-contamination with lures of different species. If trapping is used, we recommend placing at least one trap per survey site per pest; however, it is preferable to use the same number of traps as total sample sites (vineyard or vineyard block). As such, multiple traps may be necessary at each sample site and may contain a lure for one arthropod. Since the cross reactivity of the lures is also unknown, it recommended that the traps be placed at minimum 20 m apart. It is recommended that manufacturer guidelines be followed for trap set up. Specific information (where available) on type of trap, lure, replacement interval, and trap placement are given in the detailed survey tables in this document. While this method is the preferred method for most moths, if this trapping method is chosen, it is important to note that surveyors will need to make at least two trips to the vineyard; 1) to set up the trap and 2) to take samples from the trap. Lures need to be replaced at differing time intervals (from 2-12 weeks), which may add additional trips to each vineyard site.

8. The Pre-test: It is advisable to conduct a pre-test, trying the survey procedure out in several vineyards prior to deploying the procedure on a large, state-wide scale, to evaluate the survey plan to ascertain if the survey plan needs any changes.

9. The Organization of Field Work: One of the most important components of the survey is the organization of the field work. In other words, how do you organize and implement the survey to have the right resources in the correct place and data collection at the proper time? What field/vineyards are going to be sampled? What are the rows and paces to locate subsample units? What are the unique identifiers for the sample units and the subsample units? What system is setup to identify plant tissue and soil samples with sample units and subunits?

Chapter 2: Survey Design & Sampling Methodology

What vehicles, measuring tape, stakes, chaining pins, stadia rod, flagging ribbon, sample bags, traps, lures, and other equipment are needed to conduct the survey?

10. Summary and analysis of data: Good documentation is key to any future analysis. Most data will simply be entered into NAPIS and further analysis will not be necessary.

11. Gaining information for future surveys: Do you need to do something different? Is there a problem with a particular trap or with the frame? You should plan on documenting problems for use in planning future surveys.

Specific questions about this methodology may be addressed to Edward (Ned) Jones ([email protected]).

Chapter 3: Summary of Survey Strategies Section 1: Visual Survey

Field Equipment for visual surveys  Hand Lens  Vials  Quaternary  Ice chest  ______ Plastic Bags  Paper ammonia  Stakes  ______ ______ Tweezers  Pen (Disinfectant)  Flagging rib-  Field Maps bon  ______ Flashlight  Wax pencil  ______ 70% EtOH  PDA  Sharpie  List of random  Ice Packs numbers Chaining pins

Visual Survey Guide: The following is a survey guide intended to help you identify and flag diagnostic symptoms of CAPS target pests in the field. It is important to note that none of these symptoms, taken singly, are a diagnostic feature for any of the pests. In the context of the current survey, surveyors should take note of the general condition of the plant, and further examine stems, leaves, flowers, and seed/seed head for the pests of concern.

If you believe you have found a target pest, look for a more comprehensive list of symptoms in the detailed survey tables in Chapter 4. Pictures of organisms, symptoms, and evidence of organisms can be found in the Grape Commodity-based Survey Reference.

Whole Plant: First take note of the whole plant and consider whether or not the specimen is healthy. Some general “whole plant” indicators of target pests follow.

1. Basidiocarps present (rare but possible)  Phellinus noxius (Brown root rot)

2. Defoliation  Candidatus Phytoplasma australiense (Australian grapevine yellows)  Planococcus minor (Passionvine mealybug)  Phellinus noxius (Brown root rot)

3. Distortion of growth  Diabrotica speciosa (Cucurbit beetle)  Heteronychus arator (African black beetle)  Planococcus minor (Passionvine mealybug)

4. Presence of adults  Autographa gamma (Silver-Y moth)  Diabrotica speciosa (Cucurbit beetle)  Epiphyas postvittana (Light brown apple moth)  Planococcus minor (Passionvine mealybug)  Thaumatotibia leucotreta (False codling moth)

5. Presence of eggs/egg masses  Adoxophyes orana (Summer fruit tortrix)

Chapter 3: Summary of Survey Strategies

 Autographa gamma (Silver-Y moth)  Copitarsia spp. (Owlet moths)  Epiphyas postvittana (Light brown apple moth)  Eupoecilia ambiguella (European grape berry moth)  Lobesia botrana (European grapevine moth)  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (Rice cutworm)  Thaumatotibia leucotreta (False codling moth)

6. Presence of excrement or slime trails  Adoxophyes orana (Summer fruit tortrix)  Autographa gamma (Silver-Y moth)  Eupoecilia ambiguella (European grape berry moth)  Lobesia botrana (European grapevine moth)  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (Rice cutworm)  Thaumatotibia leucotreta (False codling moth)

7. Presence of larvae/immature stages  Adoxophyes orana (Summer fruit tortrix)  Autographa gamma (Silver-Y moth)  Copitarsia spp. (Owlet moths)  Planococcus minor (Passionvine mealybug)  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (Rice cutworm)  Thaumatotibia leucotreta (False codling moth)

8. Presence of pupae  Adoxophyes orana (Summer fruit tortrix)  Autographa gamma (Silver-Y moth)  Eupoecilia ambiguella (European grape berry moth)  Lobesia botrana (European grapevine moth)  Spodoptera littoralis (Egyptian cotton leafworm)

9. Stunting/less vigorous/patchy growth/yield loss  Diabrotica speciosa (Cucurbit beetle)  Eupoecilia ambiguella (European grape berry moth)  Lobesia botrana (European grapevine moth)  Phellinus noxius (Brown root rot)  Planococcus minor (Passionvine mealybug)  Thaumatotibia leucotreta (False codling moth)

10. Whole plant toppled or uprooted  Heteronychus arator (African black beetle)

11. Wilting  Heteronychus arator (African black beetle)  Planococcus minor (Passionvine mealybug)  Phellinus noxius (Brown root rot)  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (Rice cutworm)

Chapter 3: Summary of Survey Strategies

Leaves: Choose several leaves from each plant to examine.

1. Chlorotic leaves  Adoxophyes orana (Summer fruit tortrix)  Candidatus Phytoplasma australiense (Australian grapevine yellows)  Phellinus noxius (Brown root rot)

2. Deformed leaves, curling of foliage  Adoxophyes orana (Summer fruit tortrix)  Candidatus Phytoplasma australiense (Australian grapevine yellows)  Epiphyas postvittana (Light brown apple moth)  Planococcus minor (Passionvine mealybug)

3. Feeding damage (bare sections, holes, dead/damaged tissue, etc.)  Adoxophyes orana (Summer fruit tortrix) - damage on new growth as well  Autographa gamma (Silver-Y moth)  Copitarsia spp. (Owlet moths)  Diabrotica speciosa (Cucurbit beetle)  Epiphyas postvittana (Light brown apple moth)  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (Rice cutworm)

4. Leaf scars  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (Rice cutworm) - scratch marks

5. Leaves rolled/folded (protected feeding sites formed)  Adoxophyes orana (Summer fruit tortrix)  Autographa gamma (Silver-Y moth)  Epiphyas postvittana (Light brown apple moth)  Lobesia botrana (European grapevine moth)

6. Overlapping leaves  Candidatus Phytoplasma australiense (Australian grapevine yellows)

7. Petioles cut  Autographa gamma (Silver-Y moth)

8. Presence of adults  Diabrotica speciosa (Cucurbit beetle)  Epiphyas postvittana (Light brown apple moth)  Planococcus minor (Passionvine mealybug)  Thaumatotibia leucotreta (False codling moth)

9. Presence of eggs  Adoxophyes orana (Summer fruit tortrix)  Autographa gamma (Silver-Y moth)  Copitarsia spp. (Owlet moths)  Epiphyas postvittana (Light brown apple moth)  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (Rice cutworm)  Thaumatotibia leucotreta (False codling moth)

Chapter 3: Summary of Survey Strategies

10. Presence of larvae/immatures  Autographa gamma (Silver-Y moth)  Copitarsia spp. (Owlet moths)  Planococcus minor (Passionvine mealybug)  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (Rice cutworm)  Thaumatotibia leucotreta (False codling moth) - possible

11. Presence of pupae  Adoxophyes orana (Summer fruit tortrix)  Autographa gamma (Silver-Y moth)  Eupoecilia ambiguella (European grape berry moth)  Lobesia botrana (European grapevine moth)

12. Silk webbing  Adoxophyes orana (Summer fruit tortrix)  Autographa gamma (Silver-Y moth)

13. Reddening in red varieties  Candidatus Phytoplasma australiense (Australian grapevine yellows)

14. Shredded or wilted leaves  Adoxophyes orana (Summer fruit tortrix)

15. Skeletonization  Autographa gamma (Silver-Y moth)  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (rice cutworm)

16. Wilting of leaves  Phellinus noxius (Brown root rot)

Vines/Stems: Examine vine/stems

1. Bluish hue to stems of affected shoots  Candidatus Phytoplasma australiense (Australian grapevine yellows)

2. Bore holes  Copitarsia spp. (Owlet moths) - occasionally  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (Rice cutworm)

3. Branch/shoot dieback  Phellinus noxius (Brown root rot)

4. Dark mycelial mat/sleeve/crust up the base of the stem  Phellinus noxius (Brown root rot)

5. Green/rubbery shoots  Candidatus Phytoplasma australiense (Australian grapevine yellows)

Chapter 3: Summary of Survey Strategies

6. Feeding damage  Copitarsia spp. (Owlet moths)  Heteronychus arator (African black beetle)  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (Rice cutworm)

7. Patches of white mycelium are present between the bark and the sapwood  Phellinus noxius (Brown root rot)

8. Presence of eggs  Adoxophyes orana (Summer fruit tortrix)  Copitarsia spp. (Owlet moths)  Eupoecilia ambiguella (European grape berry moth)  Lobesia botrana (European grapevine moth) - on pedicels  Thaumatotibia leucotreta (False codling moth)

9. Presence of pupae  Adoxophyes orana (Summer fruit tortrix)  Eupoecilia ambiguella (European grape berry moth)

10. Ringbarking of vines  Heteronychus arator (African black beetle)

11. Stunting and unlignified shoots  Candidatus Phytoplasma australiense (Australian grapevine yellows)

12. Wilting and collapse of vines  Heteronychus arator (African black beetle)

Roots: Examine roots

1. Frass and tunneling present in soil around roots  Heteronychus arator (African black beetle)

Flowers: Flowers may or may not be present in the grape varieties that you examine on the days that you choose to survey. If they are available, examine several flowers for the symptoms and signs indicated below.

1. Abortion of flowers  Candidatus Phytoplasma australiense (Australian grapevine yellows)

2. Dark mycelial mat/sleeve/crust on surface of roots  Phellinus noxius (Brown root rot)

3. Feeding damage  Autographa gamma (Silver-Y moth)  Copitarsia spp. (Owlet moths)  Diabrotica speciosa (Cucurbit beetle)  Epiphyas postvittana (Light brown apple moth)  Eupoecilia ambiguella (European grape berry moth)

Chapter 3: Summary of Survey Strategies

 Lobesia botrana (European grapevine moth) - possible but not always evident  Spodoptera littoralis (Egyptian cotton leafworm)  Spodoptera litura (rice cutworm)

4. Presence of adults  Autographa gamma (Silver-Y moth)  Diabrotica speciosa (Cucurbit beetle)  Eupoecilia ambiguella (European grape berry moth)

5. Presence of eggs/egg masses  Copitarsia spp. (Owlet moths)  Eupoecilia ambiguella (European grape berry moth)  Lobesia botrana (European grapevine moth)

6. Presence of larvae inside or near floral structures  Epiphyas postvittana (Light brown apple moth)  Eupoecilia ambiguella (European grape berry moth)  Lobesia botrana (European grapevine moth)

7. Shriveling of flower bunches (sometimes bunches fall)  Candidatus Phytoplasma australiense (Australian grapevine yellows)

8. Silk Webbing  Epiphyas postvittana (Light brown apple moth)  Eupoecilia ambiguella (European grape berry moth) - glomerules (webbed bud clusters) formed  Lobesia botrana (European grapevine moth)- glomerules (webbed bud clusters) formed

Fruit: Fruit may or may not be present in the grape varieties that you examine on the days that you choose to survey. Examine several fruit bunches for the following symptoms and signs.

1. Bore holes, feeding damage  Copitarsia spp. (Owlet moths)  Epiphyas postvittana (Light brown apple moth)  Eupoecilia ambiguella (European grape berry moth)  Lobesia botrana (European grapevine moth)  Spodoptera littoralis (Egyptian cotton leafworm) - possible  Spodoptera litura (Rice cutworm) - possible  Thaumatotibia leucotreta (False codling moth)

2. Creates nests among clusters of fruit  Epiphyas postvittana (Light brown apple moth)

3. Presence of eggs/egg masses  Eupoecilia ambiguella (European grape berry moth)

4. Presence of larvae inside seed/fruit  Epiphyas postvittana (Light brown apple moth)  Eupoecilia ambiguella (European grape berry moth)  Lobesia botrana (European grapevine moth)  Thaumatotibia leucotreta (False codling moth)

Chapter 3: Summary of Survey Strategies

5. Premature ripening/Fruit drop  Lobesia botrana (European grapevine moth)  Thaumatotibia leucotreta (False codling moth)

6. Presence of pupae in old mummified fruit  Adoxophyes orana (Summer fruit tortrix)

Chapter 3: Summary of Survey Strategies Section 2: Trapping In general, trapping is a type of survey that involves the use of a trap to catch arthropods of concern in a specific location, rather than walking around and actively looking for arthropods. Often times, trap efficiency is increased through the use of some type of chemical or physical attractant. These attractants might be a light source, a food source, or a specific pheromone or chemical that is highly attractive to the target species.

In the context of the current survey, there are nine arthropods which can be surveyed via this method. The nine include Adoxophyes orana, Autographa gamma, Copitarsia spp., Epiphyas postvittana, Eupoecilia ambiguella, Lobesia botrana, Spodoptera littoralis, Spodoptera litura, and Thaumatotibia leucotreta. However, the commercial availability of the pheromone for Copitarsia spp. is unknown. Copitarsia spp., have traditionally been trapped via black light trapping for early detection surveys and is the CAPS-approved method for this pest.

Additional equipment for pheromone and other surveys

 Lures  Hammer  Sweep net  ______ ______ Traps  Paper  White/Beat  ______ ______ Tweezers  Wire cloth  ______ ______ Rubber  Tall Stakes  ______ ______ ______Gloves (optional)  ______ Nails

Chapter 4: Pest Tables Table 1: Pest by Affected Plant Part Chapter 4: Pest Tables

Table 4.1: Affected Plant Part—Parts that may have symptoms

Scientific Name Common Name Plant Part Affected Leaves Vines Roots Flowers Fruit

Adoxophyes orana Summer fruit tortrix

Silver-Y moth Autographa gamma

Owlet moths Copitarsia spp.

Diabrotica speciosa Cucurbit beetle

Epiphyas postvittana Light brown apple moth

Eupoecilia ambiguella European grape berry moth

Heteronychus arator African black beetle

Lobesia botrana European grapevine moth

Planococcus minor Passionvine mealybug

Egyptian cotton leafworm Spodoptera littoralis

Rice cutworm Spodoptera litura

Thaumatotibia leucotreta False codling moth

Candidatus Phytoplasma Australian grapevine yel- australiense lows

Brown root rot Phellinus noxius

Key to symbols

= Vine/Stem = Flowers = Leaves = Pest Fruit = Damages roots

Chapter 4: Pest Tables Section 2: Survey Methods

Table 4.2: CAPS-Approved Survey Methods

Survey Method Comments Available Visual Trapping Scientific Name Common Name

Adoxophyes orana Summer Fruit Tortrix

Autographa gamma Silver-Y moth Blacklight trap- Copitarsia spp. ping Owlet moths

Diabrotica speciosa Cucurbit beetle

Light Brown Apple Epiphyas postvittana Moth

European grape Eupoecilia ambiguella berry moth

Heteronychus arator African black beetle

Lobesia botrana European grapevine moth

Passionvine mealy- Planococcus minor bug

Spodoptera littoralis Egyptian cotton leafworm

Spodoptera litura Rice cutworm

Thaumatotibia leucotreta False Codling Moth

Candidatus Phyto- Australian grape- plasma australiense vine yellows

Phellinus noxius Brown root rot

Key to symbols

= Visual Survey =Trapping

30 Chapter 5: Detailed Survey Tables Adoxophyes orana

Chapter 5: Survey Methods

Scientific Name Common Name Survey Method Available Adoxophyes Summer fruit Time Frame: Adults are present from late May to late June (first orana tortrix generation), late July to early September (second generation), and October (third generation). Note: Fruit trees (Rosaceae) are the preferred host; grapes are a secondary (minor) host. Plant Part:

Age of Plant: Younger growth is often preferred

Preferred Method:

Traps:

Trap with Lure: The paper delta trap is the approved trap for Adoxophyes orana.

The lure information is provided below:

Lure Dispenser Dispenser Lure Length of Compound Load Type Abbreviation Effectiveness

a) Z,9-14:AC a) 0.09 gray rubber ADOX 12 weeks b) Z,11-14:AC mg septum c) Z,9-14:OH b) 0.01 d) Z,11-14:OH mg c) 0.01 mg d) 0.02 mg

IMPORTANT: Placing lures for two or more target species in a trap, should never be done unless otherwise noted here.

Trap spacing: When trapping for more than one species of moth, separate traps

for different moth species by at least 20 meters (65 feet).

Notes: Trap should be used with ends open. Trap color is up to the state and does not affect trap efficacy.

If a suspect is found: Take a sample of the immature and adult insect (if available). Be sure to include a sample of the host material on which it was found. Plant samples should be labeled and placed in sealable plastic bags accompanying the insect samples. Labels should indicate date, geographic location, and identity of the host plant. Place insect specimens in a small, rigid plastic container which should be included in the plant sample bag. Do not place more than one specimen in a container. Larvae should be preserved in ethyl alcohol. Label the sample and keep it in a cool, dry place until identification can be made. Care should be taken to preserve the specimen intact, as damaged, crushed, or moldy insects are difficult to identify. Species- Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

31 Chapter 5: Detailed Survey Tables Adoxophyes orana

level identification should be made by a qualified taxonomist. Submit the specimen for identification within 72 hours after collection.

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

32 Chapter 5: Detailed Survey Tables Autographa gamma

Scientific Name Common Name Survey Method Available Autographa Silver-Y moth Time Frame: Due to the migratory nature of this species, adult A. gamma gamma can be observed every month from April to November, usually peaking in late summer Plant Part:

Age of Plant: Older leaves are preferred. Only eats young leaves after destroying the old ones.

Preferred Method:

Traps:

Trap with lure: A plastic bucket trap (unitrap) (green canopy, yellow

funnel, white bucket) with dry kill strip is the approved trap for

Autographa gamma. See the plastic bucket trap protocol in Appendix

A of this document for more information. The lure information is

provided below:

Lure Dispenser Dispenser Lure Length of Compound Load Type Abbreviation Effectiveness a) 'Z,7- a) 0.99 gray AG 4 weeks 12:AC mg rubber b) 'Z,7- b) 0.01 septum 12:OH mg

IMPORTANT: Placing lures for two or more target species in a trap, should never be done unless otherwise noted here.

Trap spacing: When trapping for more than one species of moth, separate traps for different moth species by at least 20 meters.

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

33 Chapter 5: Detailed Survey Tables Copitarsia spp.

Scientific Name Common Name Survey Method Available Copitarsia spp. Owlet moths Time Frame: May be found throughout the year during the growing season.

Plant Part:

Age of Plant: Any age of plant attacked.

Preferred Method Traps:

Blacklight trapping:

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

34 Chapter 5: Detailed Survey Tables Diabrotica speciosa

Scientific Name Common Name Survey Method Available Diabrotica Cucurbit beetle Time Frame: Beetles can occur anytime during the growing speciosa season. Adult populations tend to be highest during the flowering period. Plant Part:

Age of Plant: Any age of plant attacked.

Preferred Method Visual:

In grape, adult beetles eat young leaf edges during budding, which usually does not seriously damage the host. During the blooming period, however, beetles have been observed on flowers eating the style, stigma, and eventually the ovary. Beetle stigma feeding determines flower aborting and, as a consequence, clusters show low numbers of flowers and fruits. Weedy hosts need to be controlled as beetles can also be observed feeding on and moving into grape from surrounding weeds.

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

35 Chapter 5: Detailed Survey Tables Epiphyas postvittana

Scientific Name Common Name Survey Method Available Epiphyas Light brown Time Frame: Spring to summer. National survey begins in July and postvittana apple moth run for a period of five months. Plant Part:

Age of Plant: In spring, the pest feeds on new buds; while later generations feed on ripened fruit.

Preferred Method Traps:

Trap with lure: A Jackson trap is the approved trap for Epiphyas postvittana. The lure information is provided below:

Lure Dispenser Dispenser Lure Length of Compound Load Type Abbreviation Effectiveness a) E,11- a) 0.962 gray LBAM 4 weeks 14:AC b) 0.038 rubber b) E,E,9,11- septum 14:AC

IMPORTANT: Placing lures for two or more target species in a trap, should never be done unless otherwise noted here.

Trap spacing: When trapping for more than one species of moth, separate traps for different moth species by at least 20 meters.

Notes: Color choice up to the State. Color does not affect trap efficacy. Red traps can reduce the number of non-targets (i.e., beneficial insects) caught in trap.

 See LBAM National Survey Guidelines (linked here and below) for recommended trap densities and trap areas for your state.

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

36 Chapter 5: Detailed Survey Tables Eupoecilia ambiguella

Scientific Common Name Name Survey Method Available Eupoecilia European Time Frame: Spring to summer. ambiguella grape berry moth Plant Part:

Mostly feed on flowers and fruit. Age of Plant: Adults and larvae are primarily observed when plants are flowering. Preferred Method Traps:

Trap with lure: A wing trap is the approved trap for E. ambiguella. The lure information is provided below:

Lure Dispenser Dispenser Lure Length of Compound Load Type Abbreviation Effectiveness a) cis-9- a) 0.1 mg gray EA 6 weeks dodecen-1- b) 0.1 mg rubber yl acetate c) 0.2 mg septum (Z,9-12:AC) b) dodecan- 1-yl acetate (12:AC) c) octadecan- 1-yl acetate (18:AC)

IMPORTANT: Placing lures for two or more target species in a trap, should never be done unless otherwise noted here.

Trap spacing: When trapping for more than one species of moth, separate traps for different moth species by at least 20 meters.

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

37 Chapter 5: Detailed Survey Tables Heteronychus arator

Scientific Name Common Name Survey Method Available Heteronychus African black Time Frame: Most damage by the African black beetle occurs arator beetle during the spring to early summer when the adults are most active crawling on the soil surface and again after new adults emerge in mid summer to fall. Plant Part:

Age of Plant: In grape, damage primarily occurs in the first two years after planting. After this time the vines become too woody to be damaged by the beetle. Older vines, however, may still be damaged, especially if they have been stressed. Preferred Method Visual:

Adult damage to plants typically involves chewing of the cortex of stems just below the surface of the ground. In woody vines (e.g., grape) and eucalyptus, this type of damage occurs most frequently, causes greater growth distortion, and is potentially fatal to newly planted cuttings or seedlings.

African black beetles eat the cuttings and rootlings at or just below ground level, ring bark the vine, and cause wilting and collapse.

The problem is greatest where vines have been planted onto old pasture land, especially if kikuyu (Pennisetum clandestinum) is present.

Plant Symptoms:  Stems show symptoms associated external feeding; plant damage typically involves chewing (fraying) of stems just below the surface of the ground.  Ringbarking of vines  Wilting and collapse of vines  The whole plant may be toppled or uprooted.

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

38 Chapter 5: Detailed Survey Tables Lobesia botrana

Scientific Name Common Name Survey Method Available Lobesia European Time Frame: The first flight of adults occurs in spring when daily botrana grapevine moth average air temperature is above the minimal threshold temperature of 10 °C for 10 to 13 days. The second flight period begins in summer. Plant Part:

Mostly feed on flowers and fruit. Age of Plant: The moth is observed when plants are flowering.

Preferred Method Traps:

Trap with Lure: There are two delta traps approved for L. botrana: 1) Paper delta trap (orange/ red, three-sided sticky interior); ends left open, and 2) Large plastic delta trap (red). The lure information is provided below:

Lure Dispenser Dispenser Type Lure Compound Load Abbreviation 'E,Z,7,9-12:AC 0.5 mg gray rubber LB septum

IMPORTANT: Placing lures for two or more target species in a trap, should never be done unless otherwise noted here.

Trap spacing: When trapping for more than one species of moth, separate traps for different moth species by at least 20 meters.

Note: The paper delta trap used for this species is the same type used in the pink bollworm program: orange/ red in color, with adhesive applied to all three of the interior trapping surfaces. It is not the same trap as used for gypsy moth (which has only two sticky surfaces).

Additional Resources: PPQ Website and Information UC IPM Datasheet

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

39 Chapter 5: Detailed Survey Tables Planococcus minor

Scientific Common Name Name Survey Method Available Planococcus Passionvine Time Frame: Mealybugs or physical evidence of mealybugs may be minor mealybug; encountered at any time during the growing season. Pacific mealybug Plant Part:

Age of Plant: Any age of plant attacked.

Preferred Method Visual:

Plant material is examined for the presence of live mealybugs. The insect body is distinctly segmented, yellow to pink in color, and covered with powdery wax, with the appearance of "having been rolled in flour". The presence of ants may indicate a mealybug problem.

Planococcus minor is a phloem feeder, and in general this may cause reduced yield, reduced plant or fruit quality, stunting, wilting, discoloration, and defoliation. Plant parts may be spotted, curled, or wilted. Indirect or secondary damage is caused by sooty mold growth on honeydew produced by the mealybug.

If a suspect is found: If a suspect is found: Take a sample of the immature and adult insect (if available). Be sure to include a sample of the host material on which it was found. Plant samples should be labeled and placed in sealable plastic bags accompanying the insect samples. Labels should indicate date, geographic location, and identity of the host plant. Place insect specimens in a small, rigid plastic container which should be included in the plant sample bag. Do not place more than one specimen in a container. Larvae should be preserved in ethyl alcohol. Label the sample and keep it in a cool, dry place until identification can be made. Care should be taken to preserve the specimen intact, as damaged, crushed, or moldy insects are difficult to identify. Species-level identification should be made by a qualified taxonomist.

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

40 Chapter 5: Detailed Survey Tables Spodoptera littoralis

Scientific Common Name Name Survey Method Available Spodoptera Egyptian Time Frame: Damage may occur from spring to fall, anytime littoralis cotton plants are actively growing leafworm Plant Part:

Age of Plant: Any age of plant attacked.

Preferred Method Traps:

Trap with lure: A plastic bucket trap [unitrap] with dry kill strip is the approved trap for Spodoptera littoralis. The lure information is provided below: Lure Dispenser Dispenser Lure Length of Compound Load Type Abbreviation Effectiveness a) 'Z,E,9,11- a) 1.99 laminate ECL 12 weeks 14:AC mg b) 'Z,E,9,12- b) 0.01 14:AC mg

The plastic bucket trap (also known as the Universal moth trap or unitrap) should have a green canopy, yellow funnel, and white bucket and should be used with dry kill strip. For instructions on using the trap (see Appendix A “Plastic Bucket Trap Protocol”).

IMPORTANT: Placing lures for two or more target species in a trap, should never be done unless otherwise noted here.

Trap spacing: When trapping for more than one species of moth, separate traps for different moth species by at least 20 meters (65 feet). As of June 2010, S. litura and S. littoralis lures should be placed in different traps and separated by at least 20 meters (65 feet).

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

41 Chapter 5: Detailed Survey Tables Spodoptera litura

Scientific Common Name Name Survey Method Available Spodoptera Rice cutworm Time Frame: S. litura may be detected any time the hosts are in litura an actively growing stage with foliage available, usually spring and fall. Plant Part:

Age of Plant: Any age of plant attacked.

Preferred Method Traps:

Trap with lure. A plastic bucket trap [unitrap] with dry kill strip is the approved trap for Spodoptera litura. The lure information is provided below:

Lure Dispenser Dispenser Lure Length of Compound Load Type Abbreviation Effectiveness a) 'Z,E,9,11- a) 1.76 laminate CL 12 weeks 14:AC mg b) 'Z,E,9,12- b) 0.24 14:AC mg

IMPORTANT: Placing lures for two or more target species in a trap, should never be done unless otherwise noted here.

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

42 Chapter 5: Detailed Survey Tables Thaumatotibia leucotreta

Scientific Name Common Name Survey Method Available Thaumatotibia False codling Time Frame: Fruiting. Surveys are best conducted during warm, leucotreta moth wet weather when the population of the pest increases Plant Part:

Age of Plant: Larval feeding and development can affect fruit development at any stage. Preferred Method Traps:

Trap with lure: Trap with lure. A wing trap is the approved trap for T. leucotreta. The lure information is provided below:

Lure Dispenser Dispenser Type Lure Compound Load Abbreviation a) 'E,8-12:AC a) 0.9 mg gray rubber FCM b) 'Z,8-12:AC b) 0.1 mg septum

IMPORTANT: Placing lures for two or more target species in a trap, should never be done unless otherwise noted here.

Trap spacing: When trapping for more than one species of moth, separate traps for different moth species by at least 20 meters.

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

43 Chapter 5: Detailed Survey Tables Candidatus Phytoplasma australiense

Scientific Name Common Name Survey Method Available Candidatus Australian Time Frame: Symptoms begin to appear in late spring and Phytoplasma grapevine increase in incidence until January/February. australiense yellows (AGY) Beyond this time, AGY symptoms begin to disappear as symptomatic leaves and shoots fall from the vine. Infected grapevines are less likely to show symptoms in summer than winter. Plant Part:

Age of Plant: Any age of plant attacked.

Preferred Method Visual:

Visual inspection for symptoms associated with the phytoplasma. Several of the known symptoms should be found together before suspecting a phytoplasma infection on grape. The disease appears most often in Chardonnay and Riesling grapes but has also been reported in other cultivars.

Plant Symptoms:  Yellow (chlorotic) and downward curled leaves that fall prematurely.  The chlorotic patches on affected leaves may become necrotic.  Reddening may be seen in red varieties.  Leaves of affected shoots can overlap one another.  Shoots are stunted and unlignified.  Abortion of flowering bunches early in the season has been observed.  Any time from flowering, bunches may shrivel and fall.  Stems of affected shoots often take on a bluish hue.  Late in the season, affected shoots tend to be green and rubbery.

If a suspect is found: To confirm disease, collect 10 to 12 leaves of various ages showing typical symptoms. Place the leaves between dry paper towels. Place samples in plastic bags. Double bag the samples and deliver promptly to a diagnostic laboratory.

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

44 Chapter 5: Detailed Survey Tables Phellinus noxius

Scientific Name Common Name Survey Method Available Phellinus noxius Brown root rot Time Frame: May be found throughout the year.

Plant Part:

Age of Plant: Any age of plant attacked.

Preferred Method Visual Survey:

Soil is scraped away around the collar and the main roots and the distinctive mycelial sleeve is often present. Particular attention should be paid to vines that appear wilted or dead. P. noxius tends to be a problem in cleared forests converted to agricultural land (tree farms) or in disturbed areas and surveys should be conducted in these areas. A dark brown mycelial mat or sleeve on the surface of the roots and up to the base of the stem is used reliably for field identification of P. noxius.

Plant Symptoms:  Symptoms of brown root rot are similar to those caused by other root rot pathogens: slow plant growth, yellowing and wilting of leaves, defoliation, branch dieback, and plant death.  P. noxius forms a thick, dark brown to black crust of mycelium around infected roots and lower stems. The leading edge of the crust is creamy white, glistens with drops of clear, brownish exudate.  Patches of white mycelium are present between the bark and sapwood.  As colonization progresses, white, soft, crumbly wood becomes laced with reddish strands of fungus hyphae that turn black with age.  Basidiocarps, or fruiting bodies, although rarely observed, are purplish brown bracts (conks) with yellow-white growing margins and concentric blackish zones towards the edges. The basidocarps are gray to brown on the spore-forming surface.  Unlike other similar fungi, there are no rhizomorphs.

If a suspect is found: Small pieces of infected wood (about 5 x 2 x 1 mm) from the expanding margin should be collected, bagged, and sent to a diagnostic laboratory for pathogen identification.

Key to symbols

= Pest Infests Vine/Stem = Pest Infests Flowers = Pest Infests Leaves = Pest Infests Fruit

= Damages roots

45 Chapter 6: Detailed Diagnostic Methods Adoxophyes orana

Chapter 6: Diagnostic Methods

Scientific Name, Common Name Tools Diagnostic Method Available Adoxophyes orana CAPS-Approved Method: Morphological. (Summer fruit tortrix moth) Adoxophyes orana may occur in mixed populations with other morphologically similar species, including other Adoxophyes species. Final identification is by dissection of male genitalic structures.

Microscope Required?: YES. To be certain of the presence of A. orana, it is necessary to examine morphological features under a microscope.

Mistaken Identities: Adoxophyes orana may occur in mixed populations with closely related or morphologically similar species. By their very secretive nature, leafrollers are difficult to detect. Distinguishing between males and females of adult Adoxophyes is difficult in general (Balachowsky 1966). According to Yasuda (1998), the extensive color and pattern variation of the forewing and morphological resemblance among Adoxophyes species have created difficulties in the identification of the species. A. orana very closely resembles two U.S. species, Adoxophyes furcatana and A. negundana but there are slight differences in male genitalia. Any identification should be confirmed by an appropriately trained entomologist.

Balachowsky, A. S. 1966. Entomologie appliquée à l'agriculture. Tome II. Lépidoptères. Masson et Cie Éditeurs, Paris.

Yasuda, T. 1998. The Japanese species of the genus Adoxophyes Meyrick (Lepidoptera, Tortricidae). Transaction of the Lepidopterological Society of Japan 49: 159-173.

Morphological Guides:

Bradley, J. D., W. G. Tremewan, and A. Smith. 1973. British Tortricoid moths. Cochylidae and Tortricidae: Tortricinae. Ray Society, London.

Yasuda, T. 1998. The Japanese species of the genus Adoxophyes Meyrick (Lepidoptera, Tortricidae). Transaction of the Lepidopterological Society of Japan 49: 159-173.

Eggs: Yellowish and deposited in masses. After hatching, the transparent egg shells remain present.

Larvae: Greenish with light hairs and warts. The head is light brown to yellow (sometimes somewhat spotted) as is the thoracic shield and the anal shield. The anal comb is very fine and long with light colored teeth. The thoracal legs are brown to black. The head is long and wide. Abdominal and anal legs are greenish.

Pupae: The pupae of A. orana are initially light brown but become dark brown towards the time of emergence of the adult moth. The length is between 8 and 11 mm. The posterior margin of the abdomen segments 2 to 8 of the pupae contains very small bristles.

Key to symbols

=Hand lens =Culture =Microscope =PCR/DNA approach

46 Chapter 6: Detailed Diagnostic Methods Adoxophyes orana

These bristles cannot be distinguished with a regular magnifying glass and are hence visible as a line. The bristles on the anterior margin of abdominal segment 2 are not well developed but present. Abdomen segments 2 and 3 do not have very pronounced cross- folds. The cremaster is present but not well outlined and is more wide than long. The specific fork-shape of the wing veins 7 and 8 is already visible in the pupal stage.

Adults:

Wings: A very specific characteristic of A. orana is the fork-shaped structure of the veins 7 and 8.

Forewing length: Male 10.0-11.0 mm, Female 11.0-13.0 mm. The forewing of the female is rather dull grayish brown, while in the male the coloration is brighter and is a yellowish brown. The male has a fold that extends about ½ of the length of the costa, and the fold is lined with whitish small glandular scales (Yasuda 1998).

Wingspan: Male 15-19 mm, Female 18-22 mm. Sexual dimorphism pronounced; antenna of male shortly ciliate, forewing with broad costal fold from base to about one-third, markings usually conspicuous, contrasting with paler ground color; female usually larger, antenna minutely ciliate, forewing without costal fold, with darker general coloration and less contrasting markings (Bradley et al. 1973).

Male: Ground color of forewing light grayish brown; markings dark brown suffused with ochreous; outer margin of basal fasciae poorly defined, oblique to middle; median fascia narrow, margins irregular, usually constricted at middle before emitting strong tornal spur; pre- apical spot broken and reduced, emitting a strong stria extending to the tornal area, and a second much thinner stria parallel with termen. Hindwing gray (Bradley et al. 1973).

Female: Forewing ground color grayish brown; markings essentially as in male but more subdued and often partially obsolete. Hindwing gray (Bradley et al. 1973).

Additional Resources: Mini-Pest Risk Assessment

Key to symbols

=Hand lens =Culture =Microscope =PCR/DNA approach

47 Chapter 6: Detailed Diagnostic Methods Autographa gamma

Scientific Name, Common Name Tools Diagnostic Method Available Autographa CAPS-Approved Method: Morphological. gamma (Silver-Y moth) Microscope Required?: YES. To be certain of the presence of A. gamma it is necessary to examine morphological features under a microscope.

Mistaken Identities: Present in Unites States: Autographa californica, Autographa psuedogamma, Syngrapha celsa, Trichloplusia ni

Not present in United States: Cornutiplusia circumflexa, Syngrapha interrogationis

Morphological Guides:

Carter, D.J. 1984. Pest Lepidoptera of Europe with special reference to the British Isles. Dr. W. Junk, Boston.

Emmett, B.J. 1980. Key for identification of lepidopterous larvae infesting Brassica crops. Plant Pathology 29: 122-123.

Nazmi, N., E. El-Kady, A. Amin, and A. Ahmed. 1981. Redescription and classification of subfamily Plusiinae in Egypt. Bulletin de la Societe Entomologique d’Egypte 63: 141-162.

Sannino, L. and B. Espinosa. 2000. Comparative morphological study on pupae of Plusiinae and observations on the vice-like abdominal structures (Lepidoptera, Noctuidae). Atlanta 31: 229- 243.

Egg: Hemispherical; strongly and irregularly ribbed and reticulated; whitish, blue-gray around micropyle.

Larva: Semilooper with three pairs of prolegs only. Head with dark patch below ocelli or entirely black, glossy. Body varies from green with pale erratic longitudinal markings to almost black. Length [of late instar larvae] variable, 30-40 mm. Head green, often with a conspicuous black streak extending posteriorly from ocellar region; in dark specimens, the black streak may be expanded to form a large blotch; body tapered towards head; prolegs present on abdominal segments 5, 6, and 10 only; body varying in color from yellowish green to greenish gray; dorsal line green bordered on either side by a sinuous, narrow, white line; irregular, narrow subdorsal line white; a white or yellowish white band between subdorsal and dorsal marginal lines; spiracles white, peritreme narrow, dark green or black; pinacula white, slightly raised; prothoracic and anal plates concolorous with integument; thoracic legs varying in color from greenish brown to black.

Pupa: Pale green when just formed, gradually turning darkish starting from dorsum; black just before adult emergence. Cuticle generally rugose, granulose on head thorax and appendages, smooth on the rest of body. Dorsal cephalic margin of A1-7 finely punctuate by very small papilliform reliefs. Body cephalic end squat, little prominent and flattened. Lanceolate portion of the labium long a little more than

Key to symbols

=Hand lens =Culture =Microscope =PCR/DNA approach

48 Chapter 6: Detailed Diagnostic Methods Autographa gamma

half of the total length. Prothoracic femora length, 8-10 times prothoracic femora width. Caudal end of wings and maxillae extending to caudal margin of A6. Maxillae very long, circling forewing tips. Metathoracic legs not visible. Abdominal spiracles elliptical (ratio length/width ca. 3-3.5/1), rather elevated and, on A3- 6, with the cephalic margin prominent with respect to the caudal. Vice-like structures with the caudal jowl regularly rounded and provided with uniformly distributed papilliform reliefs; cephalic jowl in the middle prominent. Semiannular structures, with 6-8 transversal linear thin ridges, of which the inferior and the superior ones are only sketched. Some papilliform reliefs are present underlying the prominent caudal margin. The area beneath the said structures is little rounded and has some papilliform reliefs. Cremaster as typical in the group, with a ratio length/width ca. 1/1 and the basal portion wide twice the apical. It is dorsally canaliculated at the base and irregularly rugose moving towards the posterior end (particularly on the swelling). Body length 17.4 ± 0.2 mm (range 16.0-18.8, No. = 34); body width (across the thorax) 5.3 ± 0.1 mm (r. 5-6.2, No. - 34).

Adults:

Head: Vertex and frons with densely brownish gray, erect hairs. Eyes naked, large, obscure, and densely lashed. Antennae filiform, brownish, about three-fourths of forewing, scape lighter than shaft; labial palpi strong, well developed and upturned with densely rough brownish scales. Tongue developed and coiled.

Wings: Adults of A. gamma can differ in appearance, depending on generation. Specimens of the spring generation are often small, with a more grayish color, and the later generations are often brownish and with a larger wingspan. Wings are 20 mm from mid-thorax to wing tip. Forewing large, dorsally with median area purplish-gray, marked with golden gamma shapes, subterminal line dentated with dark shades; orbicular and reniform spots oblique, constructed on middle; ventrally paler.

Venation: Sc reaching costal margin at about eight-elevenths length of wing; R1 from cell at about seven-twelfths length of cell; R2 from end of accessory cell; R3 and R4 stalked at about one-half way to margin, spaced distally; R5 connate basally with the stem of R3+R4; M1 free, M2, M3 Cu, proximated basally, spaced distally; Cu2 from cell at about five-sixths length of cell; 2A and 3A complete.

Male genitalia: Male genitalia with uncus well developed, hairy, and curved with hook end; tegumen elongate and moderately broad, vinculum moderately narrow; saccus well developed and elongate; valves elongated and broad apically; costa moderately sclerotized; cucullus moderately broad without corona, but with moderately large setae; clasper attached to the middle of valve far from clavus, elongate, finger-like with 6 small setae apically; clavus present, rounded apically and setose; aedeagus large, vesica moderately chitinized and armed with well sclerotized thorn-like cornutus.

Female genitalia: Female genitalia with anal lobes moderate, triangular and clothed with long setae, anterior apophysis shorter than posterior apophysis; ostium moderate, colliculum large and well chitinized, ducta bursa moderately long, tubular and somewhat chitinized; corpus bursa large, elongate and well chitinized at the entrance; ductus seminalis present near the top of the ductus bursa.

Key to symbols

=Hand lens =Culture =Microscope =PCR/DNA approach

49 Chapter 6: Detailed Diagnostic Methods Autographa gamma

Additional Resources: Mini-Pest Risk Assessment

Field Screening Aid

Simplified screening aid

Key to symbols

=Hand lens =Culture =Microscope =PCR/DNA approach

50 Chapter 6: Detailed Diagnostic Methods Copitarsia spp.

Scientific Name, Common Name Tools Diagnostic Method Available Copitarsia spp. CAPS-Approved Method: Morphological. (Owlet moths) Adults can be identified through genitalia dissections. Larvae can only be identified to genus.

Microscope Required?: YES. To be certain of the presence of Copitarsia spp., it is necessary to examine morphological features under a microscope.

Mistaken Identities: Agrotis spp., Euxoa spp., Polia spp., and Orthosia spp.

Morphological Guides:

Riley, D.R. 1998. Identification key for Copitarsia, Spodoptera exigua, and Peridorma saucia, US Department of Agriculture, Animal and Plant Health Inspection Service (Internal Report). Pharr, TX.

Simmons, R.B., and Pogue, M.G. 2004. Redescription of two often confused Noctuid pests, Copitarsia decolora and Copitarsia incommoda (Lepidoptera: Noctuidae: Cucullinae). Annual Entomology Society of America 97(6) 1159-1164.

Simmons. R.B, and Scheffer, S.J. 2004. Evidence of cryptic species within the pest Copitarsia turbata (Herrich-Shaffer) (Lepidoptera: Noctuidae). Annals of Entomology Society of America 97(4): 675 680.

Copitarsia decolora: Description. Medium-sized, light brown or gray moths with well- defined orbicular and reniform spots.

Discussion. C. decolora varies slightly in coloration from lighter to medium brown. Females tend to be larger and have darker hindwings than males. Mitochondrial DNA evidence indicates at least two morphologically cryptic species within C. decolora: one ranging from southern Mexico to Ecuador, the other occurring in Ecuador, Colombia and Peru (Simmons and Scheffer, 2004).

Diagnosis. C. decolora lacks the brush-like androconia found in male C. incommoda. Male C. decolora have a blunt digitus and corona of spines on the valve. Female C. decolora are recognizable due to the speculate, heavily sclerotized antevaginal plate.

Male. Head. Brown; antenna light brown, biserrate and ciliated; palpus light brown, apex white.

Thorax. Patagium brownish gray; mesothorax pale brown; metathorax gray to white; fore, mid, and hindleg mixed with white and brown scales, tibial spurs striped with brown; tarsi white.

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51 Chapter 6: Detailed Diagnostic Methods Copitarsia spp.

Wings. Forewing. Length = 13 to 18 mm (average = 16.1 mm, SD = 1.3 mm, n= 14). Ground color light brown or gray; antemedial and postmedial lines, double row of brown zigzag lines, with white between them; basal area with well defined brown lines; reniform spot brown outlined in white; orbicular spot ground color with white inner and black outer margin; outer margin with triangular black spots between wing veins; fringe grayish brown.

Hindwing. Ground color white; wide marginal band brown; veins toward wing margin brown; fringe brown basally, remainder white.

Abdomen. First three abdominal segments light gray, remainder of abdomen gray; genital tuft gray; sclerotized patches present in pleural membrane near second abdominal segment; hair brushes, scent pouches and modified S2 absent; terminal tergite weakly sclerotized medially, more heavily sclerotized laterally, forming two circular areas.

Genitalia. Tegumen rounded; uncus apically swollen, bearing long setae; saccus extended into narrow point; valve sinuate, tapering to pointed apex; corona present; ampulla attenuate, apex extending beyond costal margin of valve; digitus spatulate; juxta a broad plate with pointed lateral margins, medio-ventral plate with rounded, sinnuate margins, dorsal margin V-shaped with a pair of ventrally produced arms with dorsally curved apices; spinose pad present above aedeagus; apex of aedeagus with a small sclerotized plate (sp) consisting of one large and two pointed projections, a large serrate sclerotized plate (lp) opposite small plate; vesica elongate; cornuti various sized elongate spines in both clusters and solitary in a spiral line in basal one-quarter of vesica.

Female. As in male, except antennae filiform and cilated; forewing length = 14 to 18 mm (average = 16.8 mm, SD = 1.2 mm, n =24); hindwing darker than males.

Genitalia. Papillae anales, posterior apophyses unmodified; anterior apophyses reduced in length, thickened; S8 unmodified; antevaginal plate U-shaped, spiculate texture, symmetrical; ductus bursae sclerotized, spinose; corpus bursae deeply ridged, spherical, three lines of signa; appendix bursae larger than corpus bursae, membranous, irregular in shape; ductus seminalis from posterior of appendix bursae.

Copitarsia incommoda:

Description. Medium-sized, pale brown moths, with well-defined orbicular and reniform spots, and light brown hindwings.

Discussion. C. incommoda varies slightly in coloration from lighter to medium brown. Females tend to be larger and have darker

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52 Chapter 6: Detailed Diagnostic Methods Copitarsia spp.

hindwings than males.

Diagnosis. C. incommoda is often confused with C. decolora. Males of C. incommoda can be identified externally by their brush-like androconia on the second abdominal segment (sometimes only after dissection), which are absent in C. decolora. Male C. incommoda has a rounded digitus, and valves lack a corona of spines that are present in C. decolora. Female C. incommoda can be identified by the smooth texture of the U-shaped antevaginal plate, compared with the spiculate antevaginal plate found in C. decolora.

Male. Head. Brown; antenna pale brown, filiform and ciliated; palpus brown.

Thorax. Patagium brown; mesothorax lighter, tawny brown; metathorax cream to white; fore, mid, and hindleg mixed white and brown, tibial spurs striped with brown, tarsi white.

Wings. Forewing. Length = 14 to 18 mm (average = 16 mm, SD = 1.3 mm, n = 15). Ground color light brown; antemedial and postmedial lines, a double row of brown zigzag lines with white between them; basal area with well-defined brown lines; reniform spot ground color with white inner and black outer margin; orbicular spot ground color outlined in black; outer margin with triangular black spots between wing veins; fringe brown.

Hindwing. Ground color brown mixed with white scales basally; fringe light brown basally, rest white.

Abdomen. Brown, genital tuft white; hair brushes, scent pouches and modified S2 present; terminal tergite as in C. decolora.

Genitalia. As in C. decolora, except corona absent; digitus slender, apex round, not spatulate; apex of aedeagus with a small sclerotized plate (sp) consisting of one large, one small, and three minute pointed projections; a series of variously sized, heavily sclerotized spines opposite small plate (ss); cornuti in a similar pattern to that of C. decolora, but more robust.

Female. As in male, except forewing length = 14 to 19 mm (average = 17.2 mm, SD = 1.3 mm, n = 18); hindwing darker than males.

Genitalia. As in C. decolora, except lateral lobes of U- shaped antevaginal plate larger than C. decolora.

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53 Chapter 6: Detailed Diagnostic Methods Diabrotica speciosa

Scientific Name, Diagnostic Method Available Common Name Tools Diabrotica CAPS-Approved Method: speciosa Morphological- (Cucurbit beetle) Diabrotica speciosa is almost identical to D. balteata, which is widely present in the southern United States. Confirmation by a chrysomelid specialist is required.

Microscope Required? YES. To be certain of the presence of D. speciosa, it is necessary to examine morphological features under a microscope.

Mistaken Identities: D. speciosa can also be confused with Diabrotica viridula (not present in the United States) and other pestiferous Diabrotica species in South America.

Morphological Guides:

Araujo Marques, M. 1941. Contributio ao estudo dos crisomeledeos do gunero Diabrotica. Bol. Escola Nac. Agron., 2:61-143. (In Portuguese)

Baly, J.S. 1886. The Colombian species of the genus Diabrotica, with descriptions of those hitherto uncharacterized. Part I. Zoological Journal of the Linnean Society, 19:213-229.

Christensen, J.R. 1943. Estudio sobre el g'nero Diabrotica Chev. en la Argentina. Rev. Facultad de Agronomia y Veterinaria, 10:464-516. (In Spanish)

Defago, M.T. 1991. Caracterizacion del tercer estadio larval de Diabrotica speciosa. Rev. Peruana de Ent., 33:102-104. (In Spanish)

Krysan, J.L. 1986. Introduction: biology, distribution, and identification of pest Diabrotica. In: [Krysan JL, Miller TA, eds.] Methods for the Study of Pest Diabrotica. New York, USA: Springer.

Eggs: Eggs are ovoid, about 0.74 x 0.36 mm, clear white to pale yellow. They exhibit fine reticulation that under the microscope appears like a pattern of polygonal ridges that enclose a variable number of pits (12 to 30). Eggs are laid in the soil near the base of a host plant in clusters, lightly agglutinated by a colorless secretion. The mandibles and anal plate of the developing larvae can be seen in mature eggs.

Larvae: Defago (1991) published a detailed description of the third instar of D. speciosa. First instars are about 1.2 mm long, and mature third instars are about 8.5 mm long. They are subcylindrical; chalky white; head capsule dirty yellow to light brown, epicraneal and frontal sutures lighter, with long light-brown setae; mandibles reddish dark brown; antennae and palpi pale yellow. Body covered by sparse, short, dark setae; light brown irregular prothoracic plate; dark brown anal plate on the ninth segment, with a pair of small urogomphi. A pygopod is formed by the tenth segment, which serves as a locomotion and adherence organ.

Pupae: Pupae are 5.8 to 7.1 mm long and white. Females with a

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54 Chapter 6: Detailed Diagnostic Methods Diabrotica speciosa

pair of tubercles near the apex. Mature third instars build an 8 x 4 mm oval cell in the soil in which they pupate, and tenerals remain for about 3 days.

Adults: Full descriptions of D. speciosa are given by Baly (1886), Araujo Marques (1941), and Christensen (1943). Adults are 5.5 to 7.3 mm long; antennae 4 to 5 mm. General color grass-green (USDA, 1957); antennae filiform and dark (reddish-brown to black) and nearly equal to the body in length, first three basal segments lighter; head ranging from reddish brown to black; labrum, scutellum, metathorax, tibiae and tarsi black; elytra each with three large oval transverse spots, basal spots larger and usually reddish toward the humeral callus, the rest yellow. Ventrally, head and metathorax dark brown, prothorax green, mesothorax and abdomen light brown or yellow-green. Pronotum bi-foveate, convex, smooth, shiny, ¼ wider than long. Male antennae proportionally longer than female antennae. Males with an extra sclerite on the apex of the abdomen that makes it look blunt, compared with the rather pointed female apex.

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55 Chapter 6: Detailed Diagnostic Methods Epiphyas postvittana

Scientific Name, Diagnostic Method Available Common Name Tools Epiphyas CAPS-Approved Method: Morphological. postvittana (Light brown apple Identification requires dissection of male genitalia. Female moth) specimens should be sent to a Lepidopteran specialist for identification. Sorting and Level 1 Screening may be performed without dissection by using Passoa et al. (n.d.).

Passoa, S., M. Epstein, T. Gilligan, J. Brambila, M. O'Donnell. n.d. Light Brown Apple Moth (LBAM) Epiphyas postvittana (Walker) Screening and Identification Aid.

Microscope Required?: Yes. Positive identification can be made with certainty only by examining the adult moth’s reproductive organs.

Mistaken Identities: Many native tortricids could be confused with E. postvittana.

Morphological Guides:

Bradley, J.D. 1973. Epiphyas postvittana (Walker), pp. 126-127, British Tortricoid moths; Cochylidae and Tortricidae; Tortricinae. The Ray Society, London.

Bradley, J.D., Tremewan, W.G., and Smith, A. 1979. List of British Species of Tortricidae: Olethreutinae, London.

Hampson, G.F. 1863. List of the Lepidopterous insects in the British Museum. Part XXVII. Crambites and Tortricites. British Museum of Natural History, London.

Scott, R.R. 1984. New Zealand pest and beneficial insects. Lincoln University College of Agriculture, Canterbury, New Zealand.

Zimmerman, E.C. 1978. Insects of Hawaii: Microlepidoptera. University Press of Hawaii, Honolulu.

Eggs: Pale green to pale brown, broadly oval, almost flat, 0.84 to 0.95 mm. Females deposit eggs in egg masses. Within a mass, eggs are laid slightly overlapping each other like fish scales and covered in a greenish “waxy secretion”. Immediately prior to hatching, the dark head of the developing larvae is visible.

Larvae: First instar larvae are approximately 1.6 mm long, and final instar larvae range from 10 to 20 mm in length. The body of a mature larva is green with a darker green central stripe and two side stripes. The first larval instar has a dark-brown head; all other instars have a light-fawn head and prothoracic plate. The hairs on the body are whitish. The thoracic legs are the same color as the head, but paler, and are unmarked. Larvae have a greenish anal comb with seven teeth. Overwintering larvae are typically darker.

Pupae: Pupae are green after pupation but become brown within one day. Male pupae average 2.5 by 7.6 mm; females average 2.9 by 9.8 mm. The pupal stage is completed within the “nests” (thin- walled silken cocoons) made up of rolled up leaves. Pupae of all

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56 Chapter 6: Detailed Diagnostic Methods Epiphyas postvittana

tortricids are similar in appearance.

Adult: Light brown apple moth adults are highly sexually dimorphic (males are usually smaller) and variable in wing pattern and color, although a lighter, diamond-shaped area extending from behind the head to approximately one-third of body length is typically visible at rest.

Male forewing length ranges from 6 to 10 mm, compared with 7 to 13 mm in females. Males tend to have a higher contrast in coloration of the wings than females, although the level of contrast varies. Typical males have a light brown area at the base distinguishable from a much darker, red-brown area at the tip. The latter may be absent, the moth appearing uniformly light brown, as in the females, with only slightly darker oblique markings distinguishing the area at the tip of the wing. The hindwings of both sexes are pale brown to gray, either uniform in color or mottled with wavy-brown markings.

Males have an extension of the “forward” outer edge of the forewing called the costal fold, which runs the length of the wing edge. This is an expanded part of the wing that folds up over the front edge of the wing as a flap. Females do not have this costal fold.

Additional Resources: Screening Aid

LBAM ID

Mini-Pest Risk Assessment

PPQ Program Information

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57 Chapter 6: Detailed Diagnostic Methods Eupoecilia ambiguella

Scientific Name, Diagnostic Method Available Common Name Tools Eupoecilia CAPS-Approved Method: Morphological. ambiguella (European grape Identification requires dissection of the adult male genitalia; use berry moth) Brown (2009) as an aid.

Brown, J. 2009. Adult Lepidoptera Workshop.

Microscope Required?: Yes. Positive identification of E. ambiguella requires microscopic examination.

Mistaken Identities: E. ambiguella can be confused with Endopiza viteana (present in the United States).

Morphological Guides:

Alford, D.V. 2007. Pests of Fruit Crops: A Color Handbook. Manson Publishing Ltd, London. pp. 461.

Meijerman, L., and Ulenberg, S.A. 2000. Arthropods of Economic Importance: Eurasian Tortricidae.

Egg: Eggs are 0.8 x 0.6 mm; grayish brown when laid, later becoming speckled with orange.

Larvae: Larvae are up to 12 mm long; body purplish brown or yellowish brown to olive green, with large, brown and moderately conspicuous pinacula; head and prothoracic plate dark brown or black; anal plate brownish to yellowish; abdominal prologs each with 25-30 crochets.

Pupae: Pupa are 5-8 mm long; reddish brown; cremaster with a ring of 16 hook-tipped bristles and a pair of short horns dorsally.

Adults: Adults have a 12-15 mm wingspan; forewings whitish ochreous, marked with yellow ochreous and with a dark, brownish to black median fascia; hindwings gray.

Male external characteristics: Labial palpi ochreous-cream, head and thorax cream, abdomen brownish. Antenna shortly ciliate. Forewing only slightly dilated posteriorly; ground color pale ochreous-white, extensively suffused and strigulated with yellow- ochreous, costa and dorsum strigulated or dotted with black, costa narrowly suffused with fuscous-black from base to beyond middle (outer margin of median fascia); markings dark gray; basal and sub-basal fasciae obsolescent, indicated only by costal striae; median fascia conspicuous, inwardly oblique, dilated on costa, diffusely strigulated or mixed with blackish, a variable ferruginous admixture above and below median fold, sometimes extending to dorsum; pre-apical spot obsolescent, indicated by weak costal striae; apex variably suffused with blackish; a small diffuse blackish spot in terminal margin near middle; cilia pale ochreous, a dark sub- basal line. Hindwing gray, paler basally; cilia light gray, with a dark sub-basal line.

Male genitalia: Socii expanding towards base such that bases touch each other; thin narrow terminals of socii situated laterally on

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58 Chapter 6: Detailed Diagnostic Methods Eupoecilia ambiguella

broad bases. Vinculum divided ventrally, forming two sclerites. Caudal margin of valva oblique; sacculus broad at base, narrow beyond base, with short spinules on raised terminal portion; transtilla with strong median process. Aedeagus large, with one large thin cornutus and many small cornuti, arranged in three groups: 2 smaller groups and one in which the cornuti are arranged in a ring. In the latter group, the cornuti have plateshaped bases.

Female external characteristics: Forewing markings similar to those of male; hindwing more uniformly gray.

Female genitalia: Antrum very short, broad, connected to sterigma. Ductus bursae short, distal part membranous with many small denticles, anterior part sclerotized, reaching into the corpus bursae, covered with denticles. Anterior part of corpus bursae membranous.

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59 Chapter 6: Detailed Diagnostic Methods Heteronychus arator

Scientific Name, Common Name Tools Diagnostic Method Available Heteronychus CAPS-Approved Method: Morphological. arator (African black beetle) Microscope Required?: No. African beetle larvae can be identified with the naked eye, since their anal opening is horizontal, compared with a vertical opening in other species. However, microscopic detail will aid in identification and diagnosis.

Mistaken Identities: Not present in United States.: Australaphodius frenchi

Morphological Guides:

Smith, T.J., Petty, G.J., and Villet, M.H. 1995. Description and identification of white grubs (Coleoptera: Scarabaeidae) that attack pineapple crops in South Africa. African Entomology 3: 153-166.

Cumpston, D.M. 1940. On the external morphology and biology of Heteronychus sanctae Helenae Blanch and Metanastes vulgivagus Olliff (Col., Scarabaeidae, Dynastinae). Proceedings of the Linnaean Society of New South Wales 65: 289-300.

Enrodi, S. 1985. The Dynastinae of the world. Dordrecht, Netherlands: Dr. W. Junk.

Smith et al. (1995) provided detailed illustrated descriptions and a laboratory and field key to third star larvae. Cumpston (1940) also described the features of the larvae that allow H. arator to be distinguished from other species. Keys to identify adults from related species are given by Enrodi (1985).

Eggs: White, oval, and measuring approximately 1.8 mm long at time of oviposition. Eggs grow larger through development and become more round in shape. Eggs are laid singly at a soil depth of 1 to 5 cm. Females each lay between 12 to 20 eggs total. In the field, eggs hatch after approximately 20 days. Larvae can be seen clearly with the naked eye.

Larvae: There are three larval instars. Larvae are creamy-white except for the brown head capsule and hind segments, which appear dark where the contents of the gut show through the body wall. The head capsule is smooth textured, measuring 1.5 mm, 2.4 mm, and 4.0 mm at each respective instar. The third-instar larva is approximately 25 mm long when fully developed. African black beetle larvae are soil-dwelling and resemble white 'curl grubs.’ They have three pairs of legs on the thorax, a prominent brown head with black jaws, and are up to 25 mm long. The abdomen is swollen, baggy, and gray/blue-green due to the food and soil they have eaten. Larvae eat plant roots, potentially causing significant damage to turf, horticultural crops, and ornamentals. Turf is the preferred host of the larvae.

Pupae: The larvae, when fully grown, enter a short-lived pupal stage. The pupae measure approximately 15 mm long and is typically coleopteran in form (cylindrical shape), initially pale yellow, but becoming reddish-brown nearer to the time of

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60 Chapter 6: Detailed Diagnostic Methods Heteronychus arator

emergence.

Adults: Beetles are 12 to 15 mm long; shiny black dorsally and reddish-brown ventrally. The females are slightly larger than males. Males and females are readily differentiated by the shape of the foreleg tarsus. The tarsus of the male is much thicker, shorter, and somewhat hooked compared with that of the female, which is longer and filamentous. A less obvious sexual difference is in the form of the pygidium at the end of the abdomen. In the male, it is broadly rounded, and in the female, it is apically pointed. The beetle is the main pest stage.

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61 Chapter 6: Detailed Diagnostic Methods Lobesia botrana

Scientific Name, Common Name Tools Diagnostic Method Available Lobesia botrana CAPS-Approved Method: Morphological. (European Larvae can be keyed out using Gilligan et al. (2008). Identification grapevine moth) of adults requires dissection of the male genitalia; use Brown, 2009 and Passoa, 2009.

Brown, J. 2009. Adult Lepidoptera Workshop.

Gilligan T.M., Wright, T.J., and Gibson, L. 2008. Olethreutine Moths of the Midwestern United States. An Identification Guide. Bulletin of the Ohio Biological Survey, new series, Volume 16 (2), 334 pp.

Passoa, S. 2009. Screening Key for CAPS Target Tortricidae in the Eastern and Midwestern United States (males). Lab Manual for the Lepidoptera Identification Workshop. University of Maryland.

Microscope Required?: YES. To be certain of the presence of L. botrana, it is necessary to examine morphological features under a microscope.

Mistaken Identities: L. botrana can be confused with Endopiza viteana (present in the United States) and Eupoecilia ambiguella (not present in the United States).

Morphological Guides:

Bradley, J. D., W. G. Tremewan, and A. Smith. 1979. Lobesia botrana (Denis & Schiffermüller), pp. 69-70, British Tortricoid Moths Tortricidae: Olethreutinae. The Ray Society, London, England.

Castro, A. R. 1943. Fauna entomologica de la vid en España. Estudio sistematicobiologico de las especies de mayor importanica económica. Instituto español de entomologia, Madrid.

Hannemann, H. 1961. Tribus: Olethreutini Obraztsov, pp. 180-220, Die Tierwelt Deutschlands und de Angrenzenden Meeresteile. Veb Gustav Fischer Verlag.

Razowski, J. 1989. The genera of Tortricidae (Lepidoptera). Part II: Palaearctic Olethreutinae. Acta Zoological Cracoviensia 32: 107-328.

Vennette, R. C., Davis, E.E., Dacosta, M., Heisler, H., and Larson, M. 2003. Mini Risk Assessment – Grape berry moth, Lobesia botrana (Denis & Schiffernuller) [Lepidoptera: Tortricidae]. Appendix C. Mini-Pest Risk Assessment

Eggs: The egg of L. botrana is of the so-called “flat type”, with the long axis horizontal and the micropile at one end. Elliptical, with a mean eccentricity of 0.65, the egg measures about 0.65 to 0.90 x 0.45 to 0.75 mm. Freshly laid eggs are pale cream or yellow, later becoming light gray and translucent with iridescent glints. The chorion is macroscopically smooth but presents a slight polygonal reticulation in the border and around the micropile. As typically occurs in the subfamily Olethreutinae, eggs are laid singly, and

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62 Chapter 6: Detailed Diagnostic Methods Lobesia botrana

more rarely in small clusters of two or three.

Larvae: There are usually five larval instars. Neonate larvae are about 0.95 to 1 mm long, with head and prothoracic shield deep brown, nearly black, and body light yellow. Mature larvae reach a length between 10 and 15 mm, with the head and prothoracic shield lighter than neonate larvae and the body color varying from light green to light brown, depending principally on larval nourishment.

Pupae: Female pupae are larger (5 to 9 mm) than males (4 to 7 mm). Freshly formed pupae are usually cream or light brown but also light green or blue, and a few hours later become brown or deep brown.

The sexes may be distinguished by the position of genital sketches that are placed in the IX and VIII abdominal sternites in males and females, respectively. Moreover, the male genital orifice is placed between two small lateral prominences. When adult emergence is imminent, pupae perforate the cocoon, resting the exuvia fixed outwardly in a characteristic position by cremaster spines.

Adults: Adults are 6 to 8 mm long with a wingspan of about 10 to 13 mm. The head and abdomen are cream colored; the thorax is also cream with black markings and a brown ferruginous dorsal crest. The legs have alternate pale cream and brown bands. Forewings have a mosaic-shaped pattern with black, brown, cream, red and blue ornamentation. The ground color is bluish gray and fasciae brown, shaped by a pale cream border; scales lining the costa, termen and dorsum are darker than the wing ground color.

Cilia are brown with a paler apical tip and a cream basal line along the termen. The underside is brownish gray, gradually darker towards the costa and apex. Cilia and cubital tuft are grayish brown with a paler basal line.

There is no clear sexual dimorphism, but the sexes may be easily separated by their general morphology and behavior: as in the pupal stage, males are smaller than females, they have a narrower abdomen with an anal fine comb of modified scales (hair pencils), and when disturbed they exhibit movements more quick and nervous than those of females.

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63 Chapter 6: Detailed Diagnostic Methods Planococcus minor

Scientific Name, Diagnostic Method Available Common Name Tools Planococcus minor CAPS-Approved Method: Morphological and molecular. (Passionvine mealybug, Pacific Adult females can be identified by a qualified taxonomist using a mealybug) matrix of morphological characters. For molecular analysis, maintain some specimens in 95-100% alcohol for DNA analyses. Final identification will be based on morphological identification of adult female, and for new state records, followed by molecular analysis for confirmation.

Microscope Required?: Yes. To make a species level identification it is necessary to examine morphological features under a microscope.

Mistaken Identities: Immatures can be easily confused with other Planococcus species and other mealybug genera.

Morphological Guides: Cox, J.M. 1981. Identification of Planococcus citri (Homoptera: Pseudococcidae) and the description of a new species. Systematic Entomology 6: 47-53.

Cox, J.M. 1983. An experimental study of morphological variation in mealybugs (Homoptera: Coccoidea: Pseudococcidae). Systematic Entomology 8: 361-382.

Cox, J.M. 1989. The mealybug genus Planococcus (Homoptera: Pseudococcidae). Bulletin of the British Museum (Natural History) 58(1): 1-78.

Planococcus minor is a small sucking insect with a cottony appearance. Females are oval, 1.3 to 3.2 mm long. The insect body is distinctly segmented, yellow to pink in color, and covered with powdery wax, with the appearance of "having been rolled in flour" . The margin of the body has a complete series of 18 pairs of cerarii, each cerarius with 2 conical setae (except for preocular cerarii which may have 1 or 3 setae). Legs are elongate.

It is assumed that this species is identical in appearance to P. citri as follows: body oval; slightly rounded in lateral view; body yellow when newly molted, pink or orange-brown when fully mature; legs brown-red; mealy wax covering body, not thick enough to hide body color; with dorsomedial bare area on dorsum forming central longitudinal stripe (more obvious than on P. ficus); ovisac ventral only, may be 2 times longer than body when fully formed; with 17 or 18 lateral wax filaments, most relatively short, often slightly curved, posterior pair slightly longer, filaments anterior of posterior pair small, posterior pair about 1/8 length of body. Primarily occurring on foliage of host. Oviparous, eggs yellow. Surface of lateral filaments rough.

Additional Resources: Lucid tool

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64 Chapter 6: Detailed Diagnostic Methods Spodoptera littoralis

Scientific Name, Diagnostic Method Available Common Name Tools Spodoptera CAPS-Approved Method: Morphological. littoralis (Egyptian cotton It is difficult to distinguish from S. litura without close examination of leafworm) the genitalia.

Microscope Required?: Yes. To be certain of the presence of S. littoralis it is necessary to examine morphological features under a microscope.

Mistaken Identities: Present in United States: S. dolichos, S. ornithogalli, S. latifascia and other Spodoptera species.

Not present in United States: S. litura

Morphological Guides:

Brown, E.S. and C. F. Dewhurst. 1975. The genus Spodoptera (Lepidoptera, Noctuidae) in Africa and the Near East. Bulletin of Entomological Research, 65:221-262.

Pinhey, E.C.G. 1975. Moths of Southern Africa. Descriptions and colour illustrations of 1183 species. Moths of Southern Africa.

Mochida, O. 1973. Two important insect pests, Spodoptera litura F.) and S. littoralis (Boisd.)(Lepidoptera:Noctuidae), on various crops - morphological discrimination of the adult, pupal and larval stages. Applied Entomology and Zoology, 8:205-214.

Pogue, MG. 2002. A world revision of the genus Spodoptera Guenée (Lepidoptera: Noctuidae). Memoirs of the American Entomological Society 43.

Eggs: Spherical, somewhat flattened, 0.6 mm in diameter, laid in clusters arranged in more or less regular rows in one to three layers, with hair scales derived from the tip of the abdomen of the female moth. The hair scales give the eggs a “felt-like appearance”. Usually whitish-yellow in color, changing to black just prior to hatching, due to the big head of the larva showing through the transparent shell.

Larvae: Upon hatching, larvae are 2-3 mm long with white bodies and black heads and are very difficult to detect visually. Larvae grow to 40 to 45 mm and are hairless, cylindrical, tapering towards the posterior and variable in color (blackish-gray to dark green, becoming reddish-brown or whitish-yellow). The sides of the body have dark and light longitudinal bands; dorsal side with two dark semilunar spots laterally on each segment, except for the prothorax; spots on the first and eighth abdominal segments larger than the others, interrupting the lateral lines on the first segment.

Pupae: When newly formed, pupae are green with a reddish color on the abdomen, turning dark reddish-brown after a few hours. The general shape is cylindrical, 14-20 x 5 mm, tapering towards the posterior segments of the abdomen. The last segment ends in two

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65 Chapter 6: Detailed Diagnostic Methods Spodoptera littoralis

strong straight hooks.

Adults: Moth with gray-brown body, 15 to 20 mm long; wingspan 30 to 38 mm; forewings gray to reddish brown with paler lines along the veins (in males, bluish areas occur on the wing base and tip); the ocellus is marked by two or three oblique whitish stripes. Hindwings are grayish white, iridescent with gray margins and usually lack darker veins

Additional Resources: Mini-Pest Risk Assessment

New Pest Response Guideline

Screening Aid

Wing Diagnostics

Simple Field Key of Late Instars

Expanded Key to Late Instars

Final draft - Key to Spodoptera

Passoa (2009) - Slides 13-15, 45, 46

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=Hand lens =Culture =Microscope =PCR/DNA approach

66 Chapter 6: Detailed Diagnostic Methods Spodoptera litura

Scientific Name, Diagnostic Method Available Common Name Tools Spodoptera litura CAPS-Approved Method: (Rice cutworm) Morphological- It is difficult to distinguish from S. littoralis without close examination of the genitalia.

Consult appropriate keys by Todd and Poole (1980) and Pogue, (2002). To separate from other noctuids, use the key developed by Todd and Poole (1980).

Pogue, MG. 2002. A world revision of the genus Spodoptera Guenée (Lepidoptera: Noctuidae). Memoirs of the American Entomological Society 43.

Todd, E. L. and Poole, R.W. 1980. Keys and illustrations for the armyworm moths of the Noctuidae genus Spodoptera guenee from the Western Hemisphere. Ann. Entomol. Soc. Am. 73: 722- 738.

Microscope Required? Yes. To be certain of the presence of S. litura it is necessary to examine morphological features under a microscope.

Mistaken Identities: Present in United States: S. dolichos, S. ornithogalli, S. latifascia and other Spodoptera species.

Not present in United States: S. littoralis

Morphological Guides:

Hill, D.S. 1975. Agricultural insect pests of the tropics and their control. Cambridge Univ. Press, London.

Mochida, O. 1973. Two important insect pests, Spodoptera litura F.)and S. littoralis (Boisd.)(Lepidoptera: Noctuidae), on various crops - morphological discrimination of the adult, pupal and larval stages. Applied Entomology and Zoology, 8:205-214.

Pearson, E.O. 1958. The insect pests of cotton in tropical Africa. Commonwealth Inst. Entomol., London.

Diagnosis: Some males may look different from females externally, for example, most males have a yellowish forewing patch between the antemedial and postmedial lines below vein M. The orbicular spot is more solid in the male. Forewing length is 14-17 mm, and forewing background color ranges from brown to cream. Male genitalia with juxta triangulate; base of ampulla narrower than in S. littoralis; dorsal lobes of coremata almost as long as ventral lobes. Female genitalia with distal margin of ventral plate of ostium bursa a broad V-shaped notch; ductus bursae longer than S. littoralis.

Eggs: Spherical, somewhat flattened, sculpted with approximately 40 longitudinal ribs, 0.4 - 0.7 mm in diameter; pearly green, turning black with time, laid in batches covered with pale orange-brown or pink hair-like scales from the females body.

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67 Chapter 6: Detailed Diagnostic Methods Spodoptera litura

Larva: Newly hatched larvae are tiny, blackish green with a distinct black band on the first abdominal segment. Fully grown larvae are stout and smooth with scattered short setae. Head shiny black, and conspicuous black tubercules each with a long hair on each segment. Color of fully grown larvae not constant, but varies from dark gray to dark brown, or black, sometimes marked with yellow dorsal and lateral stripes of unequal width. The lateral yellow stripe bordered dorsally with series of semilunar black marks. Mature larvae are 40-50 mm. Two large black spots on first and eight abdominal segments.

Pupa: Reddish brown in color, enclosed inside rough earthen cases in the soil, 18-22 mm long, last abdominal segment terminates in two hooks.

Adult: Body whitish to yellowish, suffused with pale red. Forewings dark brown with lighter shaded lines and stripes. Hind wings whitish with violet sheen, margin dark brown and venation brown. Thorax and abdomen orange to light brown with hair-like tufts on dorsal surface. Head clothed with tufts of light and dark brown scales. Body length 14-18 mm, wing span 28-38 mm.

Additional Resources: Mini-Pest Risk Assessment

PaDIL images

New Pest Response Guideline

Screening Aid

Wing Diagnostics

Simple Field Key of Late Instars

Expanded Key to Late Instars

Passoa (2009) - Slides 13-15, 45, 46

Key to symbols

=Hand lens =Culture =Microscope =PCR/DNA approach

68 Chapter 6: Detailed Diagnostic Methods Thaumatotibia leucotreta

Scientific Name, Common Name Tools Diagnostic Method Available Thaumatotibia CAPS-Approved Method: Morphological. leucotreta (False codling moth) Larvae: specimens must be examined under a dissecting microscope preferably by a screener experienced with the arrangement of setae on Lepidoptera larvae.

Microscope Required?: Yes. To be certain of the presence of T. leucotreta it is necessary to examine morphological features under a microscope. Mistaken Identities: Cydia spp., Cydia pomonella, Cryptophlebia spp.

Morphological Guides:

Bradley, J.D., Tremewan, W.G., and Smith, A. 1979. British tortricoid moths. Tortricidae: Olethreutinae. Vol. 2. London, England: British Museum (Natural History).

Komai, F. 1999. A taxonomic review of the genus Grapholita and allied genera (Lepidoptera: Tortricidae) in the Palaearctic region. Entomologica Scandinavica 55 (Suppl.): 1-219.

Eggs: Eggs are flat, oval (0.77 mm long by 0.60 mm wide) shaped discs with a granulated surface. The eggs are white to cream colored when initially laid, then changing to reddish color before the black head capsule of the larvae becomes visible under the chorion prior to eclosion.

Larvae: First instar (neonate) larvae approximately 1 to 1.2 mm in length with dark pinacula giving a spotted appearance, fifth instar larvae are orangey-pink, becoming more pale on sides and yellow in ventral region, 12 to 18 mm long, with a brown head capsule and first thoracic segment. The last abdominal segment bears an anal comb with 2 to 7 spines. The mean head capsule width (mm) for the first through fifth instar larvae has been recorded as: 0.22, 0.37, 0.61, 0.94 and 1.37, respectively.

Pupae: Prepupae and pupae form inside a lightly woven silk and soil cocoon formed by the fifth instar larvae on the ground. Length is 8 to 10 mm and sexual determination through morphological differences on pupal case is possible.

Adult: Adult body length 6 to 8 mm, wingspan of female and male moth is 15 to 20 and 15 to 18 (mm), respectively. Body brown, thorax with posterior double crest. Forewing is a mixture of plumbeous, brown, black, and ferruginous markings, most conspicuous being blackish triangular pre-tornal marking and crescent-shaped marking above it, and minute white sport in discal area. Male is distinguished from female by its large, pale grayish genital tuft, large dense grayish white brush hindlegs, and its heavily tufted hind tibia.

Additional Resources: New Pest Response Guidelines

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=Hand lens =Culture =Microscope =PCR/DNA approach

69 Chapter 6: Detailed Diagnostic Methods Thaumatotibia leucotreta

Mini-Pest Risk Assessment

Larval Identification Aid

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=Hand lens =Culture =Microscope =PCR/DNA approach

70 Chapter 6: Detailed Diagnostic Methods Candidatus Phytoplasma australiense

Scientific Name, Common Name Tools Diagnostic Method Available Candidatus CAPS-Approved Method: A 'universal' PCR assay has been Phytoplasma developed that enables amplification of the 16S rRNA genes of australiense phytoplasmas. Digestion of these PCR products with selected restriction enzymes provides a DNA fingerprint in the form of 16S rDNA fragment patterns that can be used to determine phytoplasma identity.

Microscope Required?: No.

Mistaken Identities: Australian grapevine yellows (AGY) resembles flavéscence dorée, bois noir, Goldgelbe Vergilbung, leaf curl, berry shrivel and other grapevine diseases (tomato big bud and an uncharacterized disease also believed to be caused by a phytoplasma).

Mechanical disruption to the phloem of grape shoots can cause symptoms similar to those associated with Ca P. australiense infection. It is important to inspect symptomatic shoots for damage to the vascular tissue due to breakage, restrictions of the vascular tissue due to tendrils or string wrapping tightly around shoots, and damage to the vascular tissue by boring insects.

Morphological Guides:

Ca. P. australiense cells, like other phytoplasmas, are surrounded by a single-unit membrane, lack a rigid cell wall, and are pleomorphic in shape. When observed by transmission electron microscopy, they appear as rounded to filamentous, pleomorphic bodies with a mean diameter of 200-800 nm (IRPCM, 2004). They are sensitive to antibiotics of the tetracycline group but not to penicillin. Like other phytoplasmas, it is an obligate intercellular parasite that occurs in the phloem sieve tubes of infected plants and the salivary glands of insect vectors.

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71 Chapter 6: Detailed Diagnostic Methods Phellinus noxius

Scientific Name, Common Name Tools Diagnostic Method Available Phellinus noxius CAPS-Approved Method: Confirmation of P. noxius is via morphological (Brown root rot) identification.

A. Surface sterilized diseased root tissues are plated on potato dextrose agar amended with ampicillin and benomyl or Chang (1995) medium.

B. The cultural characteristics of the fungus are examined and compared to photos in Ann et al. (2002) and Brooks et al. (2002).

C. The Key of the Polyporaceae described by Cunningham (1965) is then used for identification of the fungus.

Ann, P.J., Chang, T.T., and Ko, W.H. 2002. Phellinus noxius brown root rot of fruit and ornamental trees in Taiwan. Plant Disease 86(8): 820-826.

Brooks, F.E. 2002. Brown root rot. The Plant Health Instructor. DOI: 10.1094/PHI-I-2002-0923-01.

Chang, T.-T. 1995. A selective medium for Phellinus noxius. European Journal of Forest Pathology 25: 185-190.

Cunningham, G.H. 1965. Phellinus. In: Polyporaceae in New Zealand. N.Z. Dep. Sc. Ind. Res. Bull. 164: 217-240.

Microscope Required?: Yes. To be certain of the presence of P. noxius using morphological features, it is necessary to examine specimens under a microscope. Mistaken Identities: P. noxius basidiocarps are sometimes confused with P. lamaensis (not known to be present in the United States), another tropical Phellinus species. P. lamaensis sporocarps have short, reddish-brown, cone- shaped cells called hymenial setae growing into their pores; however, P. noxius does not.

According to Ann et al. (1999), after plating surface sterilized diseased root tissue on potato dextrose agar amended with ampicillin and benomyl, the cultural and morphological characteristics of the fungus are examined and compared. The Key of the Polyporaceae described by Cunningham (1965) is then used for identification of the fungus. In culture, mycelia are initially white and then brown with irregular dark brown lines or patches. In addition, staghorn-like hyphae and arthrospores, but no clamp connections are commonly observed.

Chang (1995) developed a selective medium for P. noxius using malt extract agar as a basal medium amended with benomyl, dicloran, ampicillin, and gallic acid. Tergitol NP-7 was added for isolation from soil.

Bolland et al. (1984) developed a method to induce sporulation in basidiocarps of P. noxius to obtain single spore isolates.

Bolland, L., Griffin, D.M., and Heather, W.A. 1984. Induction of sporulation in basidiomes of Phellinus noxius and preparation of single spore isolates. Bulletin of the British Mycological Society 18(2):

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72 Chapter 6: Detailed Diagnostic Methods Phellinus noxius

131-133.

Morphological Guides:

Cunningham, G.H. 1965. Phellinus. In: Polyporaceae in New Zealand. N.Z. Dep. Sc. Ind. Res. Bull. 164: 217-240.

Pegler, D.N., and Waterson, J.M. 1968. Phellinus noxius, C.M.I. descriptions of pathogenic fungi and bacteria. Commonwealth Mycological Institute 195. 2 pgs.

The basidiocarp is perennial, solitary or imbricate, sessile with a broad basal attachment, commonly resupinate. Basidiocarps are not always produced in nature but can be induced in the laboratory. Pileus 5-13 x 6-25 x 2-4 cm, applanate, dimidiate or appressed-reflexed; upper surface deep reddish-brown to umbrinous, soon blackening, at first tomentose, glabrescent, sometimes with narrow concentric zonation, developing a thick crust; margin white then concolorous, obtuse. Context up to 1 cm thick, golden brown, blackening with KOH, silky- zonate fibrous, woody. Pore surface grayish-brown to umbrinous; pores irregular, polygonal, 6-8 mm, 75-175 µm diameter, dissepiments 25-100 µm thick, brittle and lacerate; tubes stratified, developing 2-5 layers, 1-4 mm to each layer, darker than context, carbonaceous.

Basidiospores approximately 4 x 3 µm, ovoid to broadly ellipsoid, hyaline, with a smooth, slightly thickened wall, and irregular guttulate contents. Basidia 12-16 x 4-5 µm, short clavate, 4-spored. Setae absent. Setal hyphae present both in the context and the dissepiment trama. Context setal hyphae radially arranged, up to 600 x 4-13 µm, unbranched or rarely branching, with a thick dark chestnut brown wall and capillary lumen; apex acute to obtuse, occasionally nodulose. Tramal setal hyphae diverging to project into the tube cavity, 55-100 x 9-18 µm, with a thick dark chestnut-brown wall (2.5-7.5 µm thick) and a broad obtuse apex. Hyphal system dimitic with generative and skeletal hyphae, non-agglutinated in the context, but strongly agglutinated in the dissepiments. Generative hyphae 1-6.5 µm diameter, hyaline or brownish, wall thin to somewhat thickening, freely branching, simple septate. Skeletal hyphae 5-9 µm diameter, unbranched, of unlimited growth, with a thick reddish-brown wall (up to 2.5 µm thick) and continuous lumen, non-septate.

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Appendix A

Appendix A– Protocol for Plastic Bucket Traps

Appendix A– Plastic Bucket Trap Protocol

Appendix A: Plastic Bucket Trap Protocol

Appendix A– Plastic Bucket Trap Protocol

Appendix A: Plastic Bucket Trap Protocol

Appendix A– Plastic Bucket Trap Protocol

Appendix A: Plastic Bucket Trap Protocol

Appendix B

Appendix B– Example of Visual Sampling Plan Output Sampling to Compute a Nonparametric (Distribution-Free) One-Sided Upper Tolerance Limit to Test that a Large Portion of Room Surfaces Does Not Contain Contamination

Summary This report summarizes the sampling design developed by VSP based on inputs provided by the VSP user. The following table summarizes the sampling design developed by VSP. A figure that shows the sample placement on the map is also provided below.

Summary of Sampling Design

Primary Objective of Design Use a nonparametric (distribution-

free) one-sided upper tolerance

limit (UTL) to test if the true Pth per-

centile of a population exceeds the

action level

Required fraction of the population 0.99 (P=99)

to be less than the action level

Required percent confidence on 95% the decision made using the UTL Method used to compute the num- Hahn and Meeker (1991, page 169) ber of samples, n (See equations below) Sample placement method Simple random sampling Calculated total number of sam- 299 ples Number of samples on map a 299

Number of selected sample areas 35 that are not rooms Total sampling area b 126458008079.67 ft2 Total cost of sampling c $46,000.00

a This number may differ from the calculated number because of 1) grid edge effects, 2) adding judgment samples, or 3) selecting or unselecting sample areas (rooms). b This is the total surface area of all selected rooms and other selected sample areas on the map of the site. c Including measurement analyses and fixed overhead costs. See the Cost of Sampling section for an expla- nation of the costs presented here.

Floor Plan Map

Appendix B– Sample Output from Visual Sampling Plan Output

Primary Sampling Objective The primary objective of this sampling effort is to make a decision whether an unacceptably large portion (fraction) of a specified surface area (target population) is contaminated above a specified action level (AL) or is otherwise defective. It is presumed that suitable actions have been identified to be implemented for either way the decision may go.

Population Parameter of Interest The population parameter of interest is the true Pth percentile of the population of contaminant concentra- tions, where 0 < P < 100, in this case, the 99th percentile (P = 99). The true Pth percentile is the value above which (100 - P)% of the population lies and below which P% of the population lies. The objective is to reject the null hypothesis if the true Pth percentile exceeds the specified action level (AL). But, the true Pth percentile will never be known with 100% confidence because all possible measurements from the population cannot be obtained. Hence the decision whether to reject the null hypothesis is made using the computed upper tolerance limit (UTL) for the Pth percentile, that is, by computing the upper 100(1-a)% confidence limit on the Pth percentile (see Decision Rule below). For the current design a is 0.05, which means that the decision will be made using the computed UTL for the 95% confidence limit on the 99th percentile.

Hypothesis Being Tested The null hypothesis (baseline assumption) is as follows:

Ho: The true Pth percentile £ AL or equivalently, Ho: Less than P% of the population < AL

The Ho is rejected if UTL < AL, in which case the alternative hypothesis (Ha) is accepted as being true, where: Ha: More than P% of the population < AL

Sampling Design Options VSP requires that the VSP user select either • simple random sampling (SRS), or • systematic grid sampling with a random start location

to determine the room surface locations at which measurements are made or samples are collected and subsequently measured. For this design, simple random sampling was used.

Decision Rule and Number of Samples, n The null hypothesis is rejected and the alternative hypothesis is accepted if the nonparametric (distribution- free) UTL for the Pth percentile is less than the specified action level (AL). The nonparametric UTL is simply the maximum of the n measurements obtained from the population of interest, where n is computed using the following equation

n = ln (α) ln (P/100)

(from Hahn and Meeker 1991, page 169). These authors discuss the statistical meaning, use, and computa- tion of nonparametric tolerance limits and the number of samples required (pages 91, 92,169, and 326).

Appendix B– Sample Output from Visual Sampling Plan Output

The following table displays the values of the input parameters used for this design:

Parameter Value

Input

P 99

a 0.05

(5%)

Confidence (1- 95%

Output n 299

Statistical Assumptions 1. Representative measurements have been obtained from a defined target population using simple random sampling or a systematic grid pattern that has a randomly selected starting location. 2. The n measurements are statistically independent, i.e., there is no spatial correlation (no spatial patterns) of contaminant levels throughout the target population. 3. The maximum of the n measurements is not an invalid value, i.e., it is not a mistake or an unacceptably certain value due to faulty sample handling, transport, treatment, storage, or measurement.

Sensitivity Analysis The sensitivity of the calculation of number of samples was explored by varying P and CL and examining the resulting changes in the number of samples. The following table shows the results of this analysis.

Number of Samples CL=99 CL=97 CL=95 CL=93 CL=91

P=91 49 38 32 29 26 P=95 90 69 59 53 47

P=99 459 349 299 265 240

P = Required Percent of the Population to be Less Than the Action Level. CL = Confidence Level (1-a) (%)

Cost of Sampling The total cost of the completed sampling program depends on several cost inputs, some of which are fixed, and others that are based on the number of samples collected and measured. Based on the numbers of samples determined above, the estimated total cost of sampling and analysis at this site is $150500.00, which averages out to a per sample cost of $503.34. The following table summarizes the inputs and resulting cost estimates. COST INFORMATION Cost Details Per Analysis Per Sample 299 Samples Field collection costs $100.00 $29900.00 Analytical costs $400.00 $400.00 $119600.00 Sum of Field & Analytical costs $500.00 $149500.00 Fixed planning and validation costs $1000.00 Total cost $150500.00

Appendix B– Sample Output from Visual Sampling Plan Output Recommended Data Analysis Activities Post data collection activities generally follow those outlined in EPA's Guidance for Data Quality Assessment (EPA, 2000). The data analysts should become familiar with the context of the problem and goals for data collection and assessment. The n data should be verified and validated before being used to test the null hypothesis. The VSP user should enter the validated and verified n data values into the VSP dialog box and click on appropriate tabs to obtain the following statistical summaries of the data. If there is strong evidence that the n data are normally distributed, the VSP user may want to use VSP to determine the number of samples, n, required to compute the normal distribution UTL and then use that UTL (rather than the nonparametric UTL) to test the null hypothesis.

Summary statistics: n, minimum and maximum of the n measurements, range of the n data, mean, median, standard deviation, variance, skewness, percentiles, and the interquartile range

Statistical Tests of Normality Assumption: Shapiro-Wilk test (if n £ 50) (Gilbert 1987), Lilliefors test (if n > 50) (EPA 2000).

Graphical Displays of the Data: Histogram, box-and-whisker plots and quantile-quantile (probability) plots (EPA 2000).

References

EPA. 2000. Guidance for Data Quality Assessment, Practical Methods for Data Analysis, EPA QA/G-9, EPA/600/R-96/084, July 2000, Office of Environmental Information, U.S. Environmental Protection Agency.

Gilbert, R.O. 1987. Statistical Methods for Environmental Pollution Monitoring, Wiley & Sons, New York, NY.

Hahn, G.J. and W.Q. Meeker. 1991. Statistical Intervals. Wiley & Sons, Inc, New York, NY.

This report was automatically produced* by Visual Sample Plan (VSP) software version 4.6d. Software and documentation available at http://dqo.pnl.gov/vsp Software copyright (c) 2007 Battelle Memorial Institute. All rights reserved. The report contents may have been modified or reformatted by end-user of software.

The Coordinates of the samples locations are also provided in VSP. An example follows:

Area: Area 50 X Coord Y Coord Label Value Type Historical 631055.6529 -64807.1884 Random 584983.0099 -36313.6935 Random 599159.2077 -91893.5971 Random 627511.6034 -63400.1022 Random 592071.1088 -34906.6073 Random 620423.5045 -82395.7655 Random 606247.3066 -53902.2706 Random 583210.9852 -72897.9339 Random 611563.3808 -44404.4390 Random

Area: Area 45 X Coord Y Coord Label Value Type Historical 535577.1217 100631.1291 Random 586546.8165 40721.0918 Random 522834.6980 64164.1499 Random 548319.5454 48535.4445 Random 516463.4861 95421.5606 Random 567433.1809 56349.7972 Random

Appendix B– Sample Output from Visual Sampling Plan Output

541948.3335 79792.8552 Random 529205.9098 43325.8761 Random 508499.4713 74583.2868 Random 533984.3187 58954.5814 Random

Area: Area 37 X Coord Y Coord Label Value Type Historical 606396.3807 104494.0744 Random 595959.9799 80333.4572 Random 575087.1785 93380.1905 Random 616832.7814 106426.9237 Random 548996.1767 84682.3683 Random 590741.7796 97729.1015 Random 569868.9782 110775.8348 Random

Area: Area 25 X Coord Y Coord Label Value Type Historical 588507.0856 9206.5621 Random 632300.0599 35792.7963 Random 574821.7811 21022.6662 Random 596718.2683 3298.5100 Random 629562.9990 12160.5881 Random 651459.4862 -29195.7763 Random 580295.9029 -2609.5420 Random 624088.8772 23976.6922 Random 645985.3644 6252.5361 Random 591244.1465 32838.7703 Random 635037.1208 -11471.6201 Random

Appendix C

Appendix C– NASS County Estimates Example NASS County Estimates CA Grapes (2002) Cumulative County Sam- County County ID Total Acres Total Cum. Fraction Cum. Sample ple Fresno 19 232,659 232,659 78.57862568 79 79 Kern 29 96,510 329,169 111.1740686 111 32 Madera 39 84,163 413,332 139.5994159 140 29 San Joaquin 77 79,218 492,550 166.3546309 166 26 Tulare 107 63,379 555,929 187.7603566 188 22 Sonoma 97 59,169 615,098 207.74419 208 20 Napa 55 49,895 664,993 224.5958077 225 17 Monterey 53 42,507 707,500 238.9521904 239 14 Santa Barbara 83 27,201 734,701 248.1391 248 9 San Luis Obispo 79 25,953 760,654 256.9045081 257 9 Mendocino 45 17,771 778,425 262.9065142 263 6 Sacramento 67 17,257 795,682 268.7349212 269 6 Riverside 65 15,386 811,068 273.9314137 274 5 Merced 47 13,783 824,851 278.5865063 279 5 Yolo 113 13,770 838,621 283.2372083 283 4 Stanislaus 99 11,396 850,017 287.0861117 287 4 Lake 33 9,423 859,440 290.2686509 290 3 Kings 31 4,518 863,958 291.7945675 292 2 Amador 5 3,696 867,654 293.0428605 293 1 Solano 95 3,632 871,286 294.2695381 294 1 San Benito 69 2,588 873,874 295.1436134 295 1 Alameda 1 2,414 876,288 295.9589216 296 1 Santa Clara 85 2,283 878,571 296.7299857 297 1 El Dorado 17 2,173 880,744 297.4638982 297 0 Contra Costa 13 1,776 882,520 298.0637273 298 1 San Bernar- dino 71 891 883,411 298.3646551 298 0 Sutter 101 859 884,270 298.6547752 299 1 Calaveras 9 578 884,848 298.8499898 299 0 Nevada 57 525 885,373 299.0273042 299 0 Santa Cruz 87 447 885,820 299.1782747 299 0 Los Angeles 37 423 886,243 299.3211394 299 0 Butte 7 265 886,508 299.4106409 299 0 San Diego 73 264 886,772 299.4998047 299 0 Tehama 103 255 887,027 299.5859288 300 1 Placer 61 165 887,192 299.6416561 300 0 San Mateo 81 142 887,334 299.6896155 300 0 Yuba 115 142 887,476 299.7375748 300 0 Humboldt 23 137 887,613 299.7838454 300 0 Ventura 111 136 887,749 299.8297782 300 0 Trinity 105 135 887,884 299.8753733 300 0 Marin 41 117 888,001 299.9148891 300 0

Appendix C: NASS County Estimate Example

Cumulative Cum. County County ID Total Acres Total Cum. Fraction Sample County Sample Shasta 89 110 888,111 299.9520407 300 0 Mariposa 43 71 888,182 299.9760203 300 0 Siskiyou 93 35 888,217 299.9878413 300 0 Tuolumne 109 35 888,252 299.9996623 300 0 Imperial 25 1 888,253 300 300 0

California Total 888,253 300 Total samples Divide by 300 samples

2960.84333 acres/sample or 2,961 acres/sample