annual progress report research supported by north

Akhtar Ali, 2014 University of Tulsa

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Annual Progress Report

Research supported by North American Strawberry Growers Research Foundation

Association

Project title: Detection of strawberry viruses in Mid-southern states

July 16, 2014

Project Principal Investigator

Dr. Akhtar Ali

Associate Professor of Plant Virology

Department of Biological Science

The University of Tulsa

Tulsa, Oklahoma74104

Tel: 918-631-2018

Fax: 918-631-2762

Email: [email protected]

Project type: Production Research / Pest Management Strategies

mailto:[email protected]


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Significance of the project

Strawberry (Fragaria ananassa) is highly nutritious fruit which is consumed as a fresh

fruit worldwide. It is one of the cash crops grown in the US in a number of states. Plant

viruses are pathogenic to strawberry crops and could reduce the yield and quality of the

produce. Before the initiation of this project in fall 2013, little information was available

about the type of viruses infecting strawberry crops in Mid-southern states. Therefore,

this project was mainly focused to determine the incidence and distribution of viruses

infecting strawberry in these states. Information obtained from this report could be

helpful for future management strategies to reduce the impact of these viruses on

strawberry crops.

Objectives

The specific objectives of the project are:

(i) To index the source materials for plant viruses in strawberry farms located in

Arkansas, Missouri, Oklahoma and Texas.

(ii) To detect and determine the incidence of viruses in the field during the

growing season

Survey and sample collection

Several surveys were conducted from May-June 2013 and March-June, 2014 during the

growing seasons of strawberry in various Mid-southern states that includes Arkansas,

Missouri, Oklahoma and Texas. More than 500 leaf samples (both symptomatic and

asymptomatic) of strawberry were randomly collected from 27 grower’s field in 17

different farms located in Mid-southern states (Table 3). All samples were stored in

individual plastic bags and were labeled. Samples were brought to the lab on ice for

further processing.

Detection of viruses by DIBA

A small amount of tissue from each sample was crushed in a plastic bag in the presence

of an appropriate buffer. Extracted sap (2 µl) was dotted on a nitrocellulose membrane

and was replicated eight times on eight different membranes. All eight membranes were

tested individually against the antisera of eight known viruses (Table 2).

The antisera of these eight viruses (Table 2) were available commercially from AC

Diagnostics or Agdia Inc. There were no antisera for a number of viruses (Table 1) that

infect strawberry plants and that is they were not tested in this study.

Symptoms in the field

Most of the strawberry plants were asymptomatic. However, some plants were showing

typical virus-like symptoms in some farms in all four states. The most common

symptoms observed on collected samples were yellowing, mottling, and stunting,

reddening and interveinal chlorosis.


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Incidence of strawberry viruses in Mid-southern states

The results obtained in this work showed that six out of eight known viruses were

detected in strawberry fields but none of the samples were positive to ApMV and ArMV.

The highest average incidence was recorded for SMYEV (8.8%), followed by TNV

(4.5%), RDBV (3.6%), RpRSV (2.95%), while the remaining viruses (TBRV and

ToRSV) was detected in less than 2% of the samples.

All six viruses were detected in Arkansas and Missouri while only five viruses

were found in Oklahoma and Texas. In Arkansas and Missouri the incidence of all 6

viruses varied from 0 to 5.6% except SMYEV which was recorded in more than 10% of

the samples collected in Missouri. Similarly the incidence of all five viruses in Oklahoma

and Texas ranged from 0-5% except SMYEV which was recorded in more than 20% of

samples collected in Texas. The high incidence of SMYEV was probably due to the

presence of aphids that were observed in strawberry fields in both Missouri and Texas.

Aphids can multiply very quickly and could infest large number of plants in a very short

period of time if proper insecticide were not sprayed.

Fig. 1 Average incidence of strawberry viruses in Mid-southern states during 2013 and

2014 growing season.

Detection by reverse transcription-polymerase chain reaction (RT-PCR)

Total nucleic acids were extracted from approximately 100 samples that showed virus-

like symptoms and were negative by DIBA against the antisera of above eight viruses.

PCR primers were designed against Strawberry mottle virus (SMoV). So far five samples

from Missouri and 7 from Texas were positive by RT-PCR to SMoV. These are the initial

results and further work is needed to identify potential new viruses.


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Detection of viruses in DIBA-negative samples

In this work only 23% of the samples (120 samples) were tested positive to known

viruses. The remaining 76% of the samples including both asymptomatic and

symptomatic were negative to the antisera of eight viruses. These results show that there

could be many more viruses in these samples if tested with the antisera of other viruses

(if available) or by RT-PCR. For example, the above RT-PCR results showed that some

samples were positive to SMoV.

Some of the representative symptomatic plants are presented in Figure. 2.

Interestingly all of these plants were negative by DIBA when tested against the antisera

of eight viruses. It is possible that these plants are infected by viruses other than tested in

this work.

Leaf tissues from some samples showing unique symptoms were stored for the

isolation of dsRNA techniques that could potentially identify other unknown viruses.

Conclusions This current work produced preliminary results and showed that various viruses do infect

strawberry plants in different locations of Mid-southern states. However, at the moment

no significant effects of virus infection on the yield of strawberry have been observed.

This knowledge of plant viruses is necessary to develop effective management strategies.

For future study, we proposed to use nucleic acid based detection method (dsRNA

isolation) that will identify both known and unknown viruses infecting strawberry plants

without the need of the availability of virus antisera or primers of a known virus.


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Table 1. Possible plant viruses that infect strawberry plants

Name of virus Acronym Genome Vector/Inoculation Antiserum available

Apple mosaic virus ApMV (+) sense ssRNA Pollen, seed Agdia Inc., AC diag.

Arabis mosaic virus ArMV (+) sense ssRNA Nematode, seed Agdia Inc., AC diag.

Beet pseudo-yellows virus BPYV (+) sense ssRNA Whitefly -

Fragaria chilonsis cryptic virus FClCV (+) sense ssRNA Unknown -

Fragaria chiloensis latent virus FClLV (+) sense ssRNA Pollen, seed -

Raspberry bushy dwarf virus RDBV (+) sense ssRNA Pollen, seed and mechanical AC diag

Raspberry ringspot virus RpRSV (+) sense ssRNA Nematode, seed Agdia Inc., AC diag.

Strawberry chlorotic fleck assoiated virus SCFaV (+) sense ssRNA Aphid -

Strawberry crinkle virus SCV (-) sense ssRNA Aphid -

Strawberry feather leaf virus NA Unknown Unknown -

Strawberry latent virus StLV (+) sense ssRNA Unknown -

Strawberry latent C virus SLCV (-) sense ssRNA Aphid -

Strawberry latent ringspot virus SLrSV (+) sense ssRNA Nematode, seed -

Strawberry mild yellow edge virus SMYEV (+) sense ssRNA Aphid Agdia Inc., AC diag.

Strawberry mottle virus SMoV (+) sense ssRNA Aphid -

Strawberry necrotic shock virus SNSV (+) sense ssRNA Thrips, pollen, seed -

Strawberry pallidosis associated virus SpaV (+) sense ssRNA Whitefly -

Strawberry pseudo mild yellow edge virus SPMYEV (+) sense ssRNA Aphid -

Strawberry vein banding virus SVBV dsDNA-RT Aphid -

Tomato black ring virus BRSV (+) sense ssRNA Nematode, seed AC diag.

Tobacco necrosis virus TNV (+) sense ssRNA Fungus, mechanical AC diag.

Tomato ringspot virus ToRSV (+) sense ssRNA Nematode, seed Agdia Inc., AC diag.

Two commercial companies: Agdia Inc, and AC Diagnostics who carry antisera for a number of plant viruses


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Table. 2. The following eight different viruses were tested by DIBA using their polyclonal antibodies

Name of virus Acronym Genus Genome Transmission

Apple mosaic virus ApMV Ilarvirus +ssRNA Pollen, seed

Arabis mosaic virus ArMV Nepovirus +ssRNA Nematode, seed and mechanical

Raspberry bushy dwarf virus RBDV Idaeovirus +ssRNA Seed, pollen, and mechanical

Raspberry ringspot virus RpRSV Nepovirus +ssRNA Nematode and mechanical

Strawberry mild yellow edge virus SMYEV Potexvirus +ssRNA Aphids and mechanical

Tomato black ring virus TRSV Nepovirus +ssRNA Nematode, seed and mechanical

Tobacco necrosis virus TNV Necroviruses +ssRNA Fungus, mechanical

Tomato ringspot virus ToRSV Nepovirus +ssRNA Nematode, seed and mechanical

The above eight viruses were selected based on the availability of their antisera with AC Diagnostics or Agdia Inc.


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Table 3. Incidence of known viruses in strawberry crops in the Mid-southern states during 2013 and 2014 growing seasons.

State Locations No. of

fields

No. of

samples

ApMV ArMV RDBV RpRSV SMYEV TBRV TNV ToRSV Negative

samples

Arkansas Damascus 3 61 0 0 4 4 4 3 3 5

Lovell 2 28 0 0 0 0 0 0 1 0

Total 5 89 0 0 4 4 4 3 4 5 65

Missouri Pierce city 2* 31 0 0 0 0 3 0 0 0

Republic 2* 29 0 0 0 2 4 1 2 2

Wright city 2* 21 0 0 0 0 4 0 0 0

Pierce city 2 67 0 0 5 2 6 0 5 0

Total 8 148 0 0 5 4 17 1 7 2 112

Oklahoma Adair 1 31 0 0 2 3 0 1 1 0

Bixby 1 5 0 0 1 0 0 0 0 0

Collinsville 1 7 0 0 0 0 0 0 0 0

Oaks 1 10 0 0 0 0 0 0 0 0

Pocola 2 21 0 0 0 1 0 0 1 0

Stillwell 2 23 0 0 0 0 0 1 2 0

Westville 1 39 0 0 4 2 1 0 1 0

Total 8 136 0 0 7 6 1 2 5 0 115

Texas Arlington 1* 43 0 0 2 0 27 0 4 1

Collinsville 2 37 0 0 0 0 0 0 0 0

Granbury 3 30 0 0 0 0 0 0 2 1

Proctor 1 24 0 0 0 1 0 0 1 0

Total 7 134 0 0 2 1 27 0 7 2 95

Total 27 507 0 0 18 15 45 6 23 9 387

%age 0 0 3.55 2.95 8.87 1.18 4.54 1.77 76

*Samples were collected in 2013 growing seasons.


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Fig. 2. Strawberry plants showing virus-like symptoms that were negative by DIBA.


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