The Worldwide Threat from Destructive Isolates of Citrus Tristeza Virus-A Review

TRISTEZA

The Worldwide Threat from Destructive Isolates of Citrus Tristeza Virus-A Review

C. N. Roistacher and P. Moreno

ABSTRACT. This paperreviews theeffects of extremely destructiveformsofcitrustristeza^virus

(CTV) which posesserious threats to citrus industries worldwide. These include Capao Bonito CTV inBrazil, navel orange stem pitting ^CTV

in

Peru, stem pitting 12BCTV foundinthe university^orchards

in Southern California, severe grapefruit^stempitting^{CTV foundin}South Africa,recentforms of CTV responsible for decline of sweet orange ^onsour orange rootstock in Florida and Israel andother severe CTViisolatesreportedfrom Spain and elsewhere. ManyofthesedestructiveCTVisolatesare transmitted by Toroptera citricidus but most can be transmitted by Aphis gossypii at relatively high levels of efficiency. The impact of recent changes ⁱⁿ aphid transmissibility and population dynamics, and the threatof movement of 7. citricidus into new regions of the worldare reviewed. The appearance and impact of newstrainsormutantsof CTV differing in pathogenic capacities or in aphid transmissibility are ^{discussed. Methods}

for

^the identification of new or destructiveisolates^{of CTVare}

also

^reviewed.

Concepts for prevention which include quarantine, eradication and education are presented. ^The immediate needistotestfor presence of CTVⁱⁿthose countries where sourorange

is

^the predominant rootstock. Also, to test for and eliminate very destructive forms of CTV, to strengthen quarantine laws and regulations, and to educate scientists, nurseryman andgrowers^tothe dangers ^{involved in} budwood importation and virus or vector spread.

Tristeza, caused by the citrus tristeza virus(CTV) ^remains

today

^as

one ofthe most destructive ^of

all

^dis-

eases ^of citrus.

It

is an international disease recognizing no borders. The potential forspread ofsevere ^{forms of} thevirus, andthe movement ofitsprin- cipal vector, Toxoptera citricidus, into new areas, remainsaconstant threat.

The destruction _{of many} tens of ^mil- lions of trees by tristeza in the 1930's

through the ^1950's was amajor incen- tive for the formation ofthe Interna- tional Organization ofCitrus Viroloists (IOCV). Publications ontristeza areⁱⁿ the thousandsandpredominate^ineach oftheeleven proceedings oftheIOCV, signifying its importance and the _ex- tent of worldwide interest and re-

search.

There are still many

citrus

^produc-

ingcountries ⁱⁿthe Mediterranean re- gion, ⁱⁿ Central America, ⁱⁿMexico or in the Caribbean Islands where the predominant rootstock^issour orange.

The citrusindustries ⁱⁿthese countries face a real threat from the potential

ravages ^of tristeza once it begins to spread. Whenevera search is made ⁱⁿ countries where citrus is successfully grown ^onsour orange rootstock, CTV is usually found. The amount of CTV found is in direct proportion to the energy put ^into the search. Unaware growers and nurserymen

traveling

^a-

broad ^continue

to

^bringin foreign bud- wood. Some of these introduced cul- tivars contain severe CTV isolates which eventually can be transmitted efficiently by ^local aphids (6). In California andIsrael,a continuing bat- tleisbeing fought to keepcertain citrus growing areas free^oftristeza_{by inten-} sive tristeza eradication programs. ^In many countries ^where

tristeza

is now endemic, exotic and destructive CTV isolates areaconstant

threat

and some ofthese_candebilitateand evendestroy the citrus industry regardless ^of the rootstock used. Also, many countries where

7.

^{citricidus is}not present

face

the real threat ofits possible introduc- tion.

The objective ofthis review

is

^to

report the ^serious

threat

posed by new and severe

isolates

of CTV; to review changes

in

transmissibility^ofthe virus and to review some of the _{new tech-} niques for detection of CTV. Also, to re-emphasize the importance for strengthening quarantine regulations and to encourage campaigns toeducate and inform the public; especiallythose

inthe citrus industry^onthismost seri- ous threat.

NEW DESTRUCTIVE ISOLATES OF CTV.

Several severe ^and potentially ^de- structive isolates of CTV have been reported. ^Some attack the scions ^di- rectly^andare not dependenton sensi- tive rootstooks. Some isolates may be new to a region and canseverely affect thebud ^unions

of

^{citruson CTV-sensi-}

tive rootstocks. Examples

are:

— Grapefruit stem pitting ^{isolates in} South Africa (15, 40), Australia

(14), and Japan ^(42).

— TheCapao Bonitoisolate attacking sweetorangein Brazil (12,45,46).

-- Thesevere12-Bsweetorangestem pitting ^isolate reported ^from California (11, 57).

-- The sweet_orange stem pitting^iso- late destroying navel orange pro- duction in Peru (59).

-- Recently appearing destructive isolates of CTV affecting sweet orange^onsour orange ^{rootstock in} Florida(10).

-- A recent development of a new

CTV

isolate

destroying treesⁱⁿIs- rael (8, 9).

-—- Anewstempittingand destructive isolate affecting navel oranges ⁱⁿ Australia. (This outbreak ^is very recent and occurred in 1990, after presentation ^of

this

^review).

In South Africa, grapefruit is^diffi- cult to grow free ofstem pitting, ^small fruit size and tree decline. Da Graca (15) reported that the problem of de- cline ofgrapefruit associated withstem pitting^iswidespread. ^“Because

of

^this

declinethere arefew productivegrape- fruit treesin South Africaover²⁰years

Eleventh IOCV Conference old and the industry ^{has had} to gear itself to this phenomenon

by

^{the regu-}

lar removal of declining trees and the replanting ofentire ^orchards

after

they reach ¹⁶to²⁰yearsⁱⁿage”. One puzzl- ing development was the bringing ^of trees _{of a} registered Redblush grape- fruit _{into one}areaⁱⁿNatal where they developed severe stem pitting. Trees from the _same parent propagated ⁱⁿ other areas showed few symptoms.

Climatic differences may be afactorin

this differential symptom develop- ment.

At the variety collection in Kuchinotsu, Japan, several trees ^of virus-free Marsh grapefruit imported from California were grown at the ex- periment station. During ^the

first

^few

years, the fruit was normal and large sized. However,

after

the third fruiting year, the fruitwas considerably smal- ler. Many fruit _{were not} larger than small lemons and most werevery^small (Fig. ^1). Apparently, ^local severe ^iso- lates of CTV were transmitted by 7.

citricidus to these virus-free grape- fruit, inducing the smaller sized fruit.

In the extensive variety collection at the Citrus Research Station at Riverside, California, yearly surveys between 1970 and 1977 showed dieback, decline and death _{of many} trees. Many of these were seedling trees. The number of declining trees increased exponentially eachyear ^and indexing showed presence of CTV-SY isolates in many trees showingbranch dieback. This suggested that seedling yellows tristeza _was spreading ⁱⁿ the variety ^{collection (54).} At this _same time a very severe form of CTV was discovered spreading in field 12B

near the variety ^{collection (11). This}

12B” CTViisolate was found toseverely stempit and stuntall inoculated sweet orangevarieties. Transmission studies with A. gossypii showed the ^12B

iso-

late was ^100% transmissible (55, 56).

Also, when used as challenge inoculum in a cross protection experiment to sweet _orange, there was

no

^protection

by ¹⁸ of20 California field isolates of CTV(61). Becauseoftheserious poten- tial fordestruction ^of

this

and possibly

Tristeza

other seedling yellows orstem pitting isolates, an extensive indexing pro- gram ^was undertaken to

test

^{for and} destroy ^all severe forms of CTV inthe Citrus Research Station orchards.

In Capao Bonito County, San Paulo State, Brazil. asevere CTV isolate was discovered by Muller ^et al. (45, 46).

“This particular strain ^induced stunt- ing, stem pitting and poor and some- times misshapened

fruits

on practically all sweet orange scions budded on

sweet _orange or Rangpur ^{lime root-} stocks. Mostsweet_orangecitrus types previously had been considered toler- ant to the commonly occurring CTV strains ⁱⁿ Brazil”.

It

^is believed that the Capao Bonito strain was intro- duced from Japanor East Asia (60).

In Peru, navel oranges grown primarily on rough lemon rootstock were a profitable crop domestically and for export. ^Navel orange trees ^have been decelining since the 1960s. In a consultaney visit to the citrus produc- ing regions of Peru in August ¹⁹⁸⁸ under the sponsorship ofconcerned

cit-

rus growers ^and nurserymen, the senior author

visited

^the major citrus growingregionsin thecountry. Declin- ingtreesof navel orange showing small crops ofvery^smallfruit were^observed

in all coastal citrus _growing areas ^and

in all ofthe orchards

visited.

^Growers

were ⁱⁿ the process of pulling large blocks of declining navel orange trees and replanting them with mandarins.

Leaves were curled (Fig. 2a) and

often

mottled. When peeled, stems ^of

all

sizes were ^often severely pitted. ^The pits were very ^small but extremely abundant (Fig. ^2b). The bark _was somewhat thick and cheesy and stems would easily break at a node when lightly bent. Severe pitting was ^ob- served in the wood of navel, Valencia and tangelo scions and on citrumelo rootstock. Insome

locations,

new plan- tings ^ofsatsuma mandarin were adja- cent to navel orange orchards. These satsumas were propagated and in- creased from budwood imported from Japan. Importations of budwood from Japan by ^local growers was common and had been going on foryears.

The symptoms

of

stem-pitting^and small fruitsize foundin Peru appeared similar tothe symptoms

of

^{Capao Boni-}

to CTV-infected trees seen ⁱⁿ Brazil.

Also, the _easy breakage due to exces- sive stem pitting and the _{puffed and} cheesy bark were the^same

as

^{that ob-}

served ⁱⁿ sweet orange seedlings ^in- oculated with the 12B isolate found in California. Indexingofthe Peruisolate at the USDA facilities at Beltsville, Maryland showed exceptionally severe reactions _on indicator plants ^(26).

Reactions were comparable tothoseof the Brazil Capao Bonito and the

California 12B isolates.

In Florida, Brlansky etal. (10) re- ported the decline of sweet _orange trees onsour orange rootstock^{in a100} sq. ^mi. area west ^of Ft. Pierceand ⁱⁿ a ²⁰⁰ sq. ^mi. area south of Labelle.

Losses

greater

^{than 50% occurred in}

some plantings. Theyreported thatse- vere isolates of CTV causing quick de- cline andstunting werebecoming more abundant in Florida. In the LaBelle area, ^{90% of} the _young trees were found infected with a severe stunting isolate.

It

^is apparent ^fromthis report andothers that newisolatesoftristeza are posing a serious

threat

to citrus plantings on sour orange rootstock in many areasin Florida.

Inavisit

toIsraelin 1988,thesenior author observed destruction and de- cline of Shamouti orange, ^Minneola tangelo andOrtanique tangor trees ^on sour orange rootstock ⁱⁿ the Morasha Junction area near Tel Aviv. This de- cline, was first noted ^{in 1985.} Israeli scientists reported erratic results ⁱⁿ their early^index tests for detectionof CTV.

(8,

^{9). The} destruction ofcitrus by this Morasha CTV isolate was re- miniscent of the description of the deathof millions ofsweet_orange trees

on sour orange rootstock reported by Knorr and Ducharme in Argentina ⁱⁿ

1951 (35). The decline observed in Is- rael was devastating with few surviv- ing trees and appeared tospread at a rapid rate. An unusual aspect ^ofthis decline was the apparant ^{absence of} CTV in many declining trees. In ELISA and biological tests, ^{Ben Zeev}

10

et al. (9) reported many negative re- sponses even with ten samples from around each tree. Later findings by these workers suggested thata unique isolate of CTVmight

be

involved. Pos- sibly, the virus or some toxin moved rapidly from ^the

infection

^{site to the}

bud union area and induced death of the tree ^before the virusbecame

dis-

tributed in the tree. In indexing tests to lemon and sour orange, the virus was noted as a seedling ^yellows

tris-

teza. Another factor relating to the rapid spreadoftristezaⁱⁿthe Morasha Junction area was probably cultural.

The practice of heavy pruning ⁱⁿ the winter months produced a vigorous young leafflush in the spring ^{and re-} sulted in a proliferation ofaphids

(7).

Thus,

in

Israel there is now a new and very destructive type^of

tristeza

^which

can ^killatreeonsour orangerootstock even before the virus _{can be} detected by rapid indicator methods.

FACTORS WHICH CAN

INFLUENCE EPIDEMIOLOGY OF TRISTEZA

The introduction and potential movement of Toxoptera citricidus.

The potentialfor rapid movement and spread ^{of 7.} citricidus was described by F. Geraud, an entomologist ⁱⁿ Ven- ezuela who conducted anextensivesur- vey for this aphid from ¹⁹⁷⁴ to 1981.

In a personal communication hewrites

“the rapid movement of this vector from oneareatoanotherwas observed in Venezuela in 1976to ¹⁹⁷⁸by myself and collaborators. 7. citricidus _was first found in the southern Lake Maracaibo region of western Ven- ezuela in the Andes foothills in early

1976, apparently spreading from ^Col- ombia where

the

^vectorwas endemic.

Atabout the same ^time,

the

^{aphid was}

also found localized near the ^Brazilian border in southwest Venezuela.

Within this two-yearperiod the vector moved throughout Venezuela and could be detected almost everywhere

in the country^where

citrus

was grow- ing. ¹feel it wascarriedlong distances by the upper winds and efficiently

Eleventh TOCV Conference

found citrus relatives to feed on and establish colonies. ^The

first

^dramatic

tristeza disease outbreaks were ^ob- servedwithin twoyearsof colonization by 7. citricidus in the north-central region of Venezuela. There, evidence was found ofscattered small endemic fociof diseased trees thathadsurvived for about 20 years ^(without incurring decline) previous

to

^the colonization of the area by this vector.”

Since

the

first appearance^{of 7. cit-} ricidus ⁱⁿ Venezuela, over ⁶ million trees on sour orange rootstock have died from tristeza disease (41). Asimi- lar very ^rapid spread ^of this _aphid throughout Argentina was described

in_(24).the early ^1950s by ^Ducharme

et ^al.

Thereisno reason why 7.citricidus could not flourishinanycountry where citrusis grown. The aphidexistsⁱⁿ the colder regions ofJapan,

in

the subtrop-

ical regions of South America, and ⁱⁿ the dry regions of South Africa and Australia. Climatic conditions ⁱⁿ the citrus growing regions ofthe Mediter- ranean, Central ^America, the Carib- bean Islands, Mexico orⁱⁿthe USA do not differ from many regions ^where

7'.

citricidus is now endemic. With mod- ern air travel^{and the}

ability

^{of 7. cit-}

ricidusto moveacross

large

^distances

in arelatively shorttime (as shown for Venezuela or Argentina) there ^is the ever continuing danger

that

^thisaphid

will show up ⁱⁿ new areas. The aphid isnowreportedin ^Panama,

Costa

^Rica, El Salvador and Honduras (36). If 7.

citricidus becomes wellestablished in

Central America, it_{may be}justa mat-

ter

of time before it will spread north to Mexico and to the United States.

Not only isthisaphid the primaryvec- tor _{for many} severe isolates of CTV but it _{is a} major pest ^of citrus. The danger ^from the introduction of this serious pestby

airtravelisa

continuing concern.

Changes in transmissibility of CTV. Bar-Joseph and Loebenstein (5)

first showed achange

in

transmissibil- ity of CTV by A. gossypii from ^2-5%

in existing isolates to 40.7%

ina

^CTV-

SY isolate in the HibetZion region of

Tristeza

Israel. In California, comprehensive transmission tests by Dickson ^et al.

(17) in the 1950s with A. gossypit showed atransmission ratefor CTV of not more than 5-6%. This has now changed dramatically. In extensive transmission studies between 1976-79,

transmission rates were shown to be at ^100%for alarge numberof CTV and CTV-SY isolates obtained from trees

in southern and central California (55, 56). This change intransmissibility ^of CTV was probably responsible forthe spread^{of CTV-SY and}

the

decline and deathoftrees ⁱⁿthe university variety collection at Riverside. Recent studies in Spain and ⁱⁿ Florida have shown relatively high transmission rates of CTV by^A.gossypii. Hermoso de Men- dozaetal.(28) tested two CTV isolates in Spain and found transmission rates of28and 78%. Studies by ^Yokomi and Garnsey⁽⁶³⁾ with four CTV isolates in

Florida showed transmission rates of CTV by A. gossypii of¹¹ to ^53%.

A.

citricola

was ^shown

to

^{be a vec-}

tor for CTV in Florida, Spain and Is- rael, but not ⁱⁿ California. Early studies with this aphid ⁱⁿ Florida showedit tobe arelatively_poorvector for CTV (47). Studies by Yokomi and Garnsey (63) showed ¹¹ of38^CTV

iso-

lates were transmitted by^A.

citricola

compared to ²⁹ of 38 by A. gossypii.

Comparative studies ⁱⁿ Florida with four CTV isolates transmitted by A.

gossypit and A. citricola, showed transmission rates of 17.9 and 6.3%

respectively(63). In a similar compari- son with T-300, a common Spanish ^iso- late,_obtainedHermoso de_78%transmissionMendoza

et

with A. yos-^{al. (28),} sypi

but

^{only6% withA.}

^citricola.

^In

the citrus areaof Valencia (Spain),

4.

citricola _was about ⁴⁵ times more abundant thanA. gossypii but only¹³

times

less efficient

ⁱⁿ transmitting the CTV isolate T-300 (29). The rate of CTV spread achieved by vectors ⁱⁿ a given citrus areawill depend not only onthetransmission

efficiency

^{of aphid}

species present but^alsoon^the

relative

abundance

of

each species. Although A. gossypii is a more efficient vector then^A.

citricola,

population dynamics

11

must be considered. ^A.

citricola

^is far more abundant than A. gossypit.

Yokomi (personal communication) re- ports: ^“in Florida the Spirea aphid is extremely abundantand exhibits much alate _activity. This is of great epidemiological importance, even thoughit hasalowvector efficiency”.

Variations in the balance of aphid

fauna.

Changes ⁱⁿ the composition of the aphid fauna may also occur as a consequence

of

several factors such as selective resistance to certain _pes- ticides, variationsinthe population of natural enemies or changes favoring

population increase^of

efficient

vector species may have implications ⁱⁿ the movement ofCTV. Anexample^{of vari-} ation in the balance of aphid fauna seems to have occurred in the citrus area ^{of La} Plana (Spain). ^A survey ⁱⁿ

1986-87 showed

that

^{A. citricola ac-}

counted for 40-58% ofthe total _aphid population and A. gossypii for ^35-50%

(30). Previous surveys ^{in 1974-78,}

1980-81, and 1984-85 showed that A.

citricola _was about 90% of the total aphid population and A. gossypii less than ^2%. Consequently the great ^dif- ference in vector efficiency between both species plus theaphid population dynamics ⁱⁿ La Plana couldaccelerate CTV spread ⁱⁿ this citrus area.

Introduction of^{exotic CTV}isolates and increase in vector transmissibil- ity. Severe CTV isolates have been introduced into many countries but have remained quiescent for lack of transmission. This was certainly true at the University of California variety collection in Riverside where known CTV and CTV-SY isolates were pres- entin theoriginal introductions as cut- tings or grafted scions. Between 1910 and 1963, trees on sour orange rootstocks adjacent to CTV-infected introductions did not become infected (57). CTV isolates were known to be present ⁱⁿ Spain and ⁱⁿ Israel 25 yr before outbreakofthe disease. There are reports ^from Texas (48), Turkey

(3), Italy ⁽¹³⁾ and from Iran (25) of CTV-infected trees surrounded by trees on CTV susceptible rootstocks whichshowedlittle_ornoinfection. The

12

presence ^of these quiescent ^{CTV iso-} lates representareal dangerifefficient vectors should move ⁱⁿ or changes should occur ⁱⁿ their transmissibility.

Examples

illustrating

^{both situations}

are the movement ofCTV ⁱⁿ Venezuela following the introduction of 7. cit- ricidus andthe spread^ofsevere^CTV- SY by A. gossypii at the University^of California variety collection ⁱⁿ River- side. These examples should encour- age the implementation of intensive programs

for

^{CTVdetectionand eradi-}

cation of infected trees

in

citrus areas where the incidence of virus is low.

Potentially destructive isolates should be indentified in areas where ^{CTV is} already ^endemic.

Once a new ^CTV

isolate

begins to spread by vector it _{may be} very ^diffi- cult to prevent an epidemic. The dynamics and equations ⁱⁿ temporal spread of CTV by aphids, and the re- lationship of aphids and CTV are com-

prehensively reviewed by Raccah etal

(52) and Bar-Joseph ^et al. (7).

Certain cultural practices _may ^in- crease the rate ^of natural spread ^of CTV by aphids,

e.g.,

^{heavy pruningof}

citrus trees and topworking ^to other cultivars, common practices ⁱⁿ Spain andIsrael. Severely pruned trees pro- duce a vigorous young

leaf

^{flush inthe}

spring resulting ⁱⁿ a proliferation of aphids. This factor was probably re- lated to the rapidspread^{of CTVin} the Morasha Junction area ⁱⁿ Israel and has been aprimary factorⁱⁿ the intro- duction andspread ofthe virusⁱⁿmany citrus areas in Spain.

Possible change in virulence ofthe virus andthe invisible virus. Thepre- sence of different strains or mixtures of strains of CTV hidden in tolerant varieties and the eventual appearance or segregation ofmutants differing ⁱⁿ their pathogenic capabilities or trans- missibilities may beparamount to un- derstanding the epidemiology oftriste-

za. In most cases CTVexists_symptom- less insweet orange or mandarin.

Itis

conceivable that CTV, as with other viruses, does not have a repair mechanism to correct transcriptioner- rors during the replication. Thus, any

Eleventh IOCV Conference

change or mutation occurring during replication ^will remain and accumu- late. Since CTV has ^one

of

the largest

genomes

of

any virus, acertain number of errors are likely to occur and these punctual mutations _{may be} undetect- able or result ⁱⁿ certain changes ⁱⁿ biological orbiochemical properties.

Biological variability for CTV is well documented. Large variations exist ⁱⁿ symptom expression ^on field trees as well as ^on indicator plants.

Changes ⁱⁿ aphid transmissibility as occurred in California andinIsrael are also examples of variability of CTV Within a host. ^Raccah,

et

^{al.(51)showed}

segregation of isolates of CTV with variable transmissibility ^and suggest- ed “that tristeza-infected trees could harbor _more than_onevariantand that trees from a location where limited tristeza spread is observed could con- tain CTV variants that are highly transmissible but are quantitatively suppressed by a dominating poorly transmissible variant”.

Biochemical variability ^is perhaps less documented due to inherent dif- ficulties. However thereis_somerecent evidence of differing ^dsRNA

patterns

in plants infected with CTV isolates having similar biological properties

(43, 44). Differences ⁱⁿ peptide maps of^CTV

isolates

having similar biologi- cal properties and the same dsRNA patterns ^{(27) is} another indication of this variability.

Direct evidence has been obtained for the presence^{within an}

isolate

^from

a single source of several

virus

^strains

differing ⁱⁿ serological properties orⁱⁿ the dsRNA pattern ^{induced on} host plants. ^Kano

et

^{al. (34) observed that}

some aphid-inoculated CTV-infected plants failed to react with the strain- specific monoclonal antibody MCA-13 even thoughthe source ofinoculum did react withthissame antibody. Moreno etal. (44) obtained segregation^{of vari-} ants having different ^dsRNA

profiles

from a single source by graft-inoculat- ingseveral citron plants ^with bark in- oculum from a lime plant recently ^in- oculated by aphids. Both findings suggested the coexistence ofdifferent

Tristeza

CTV mutants irregularly distributed

in the source plant.

Under

these

circumstances it is not difficult to conceive natural segrega- tion ofmutantsby aphidtransmission, by budwoodpropagationor by changes in the balance of variants present within a plant, due to changes ⁱⁿ the environmental conditions. Thus, the

“invisible virus” present ⁱⁿ symptom- less ortolerant hostsmay show up and give rise _{to new} severe strains or to highly transmissible severe variants when propagated as new

varieties.

These strains _{may be}vector transmit- ted at high rates ^ofefficiency or show new and extensive _symptoms under different environmental conditions.

DETECTION PROCEDURES FOR IDENTIFYING SEVERE CTV-SY AND CTV-SP ISOLATES

Due

to

^{thewide biological}

^diversity

of CTV, any strategy to control dam- age caused by severe variants ^ofthis pathogen requires accurate proce- dures

to

identify virus strains. At the present time, theonly consistently re- liable method for identification of se- vere ^{CTV-SY and CTV-SP}

isolates is

by biological indexing. This is avery costly andtime consuming process and new technology ^is urgently ^{needed to} replace plant indexing. The California seedling yellows eradication program

isone example of anintensive program designed forthe detectionand eradica- tion of severe CTV-SY isolates using biological indexing. Over 20,000 trees

in the Citrus Experiment Station or- chards were indexed forthe presence of both CTV-SY and severe stem pit- ting isolates using grapefruit, sour orange ^and sweet orange indicators.

The procedures ^involved

the collecting

and labeling of budsticks and graft-in- oculation to grapefruit seedlings.

These were held for ^3-4 months for observation and reading for seedling yellows andstem pitting. ^{Positive con-} trols were always included. Budwood from all infected and suspect trees were recollected and further inocu- lated tosour orange andsweet _orange

13

seedlings to determine the severity ^of the CTV-SY isolate. Seedlings re- quired ^9-12 months of care from seed plantingto inoculation, andanother3-4 months for reading after inoculation.

Although this ^is a difficult and long term_process,

it

^{was the}only available andsecure technology fordetermining the presence ^ofsevere ^CTV

isolates.

In addition to biological testing, there is currently new technology for differentiating the more severe ^from the _non-severe reacting CTV isolates.

The four techniques cited below de- pend ondifferent properties ^ofthe

vir-

us and offer some promise for strain identification.

Analysis of dsRNA in ^{CTV in-} fected plants. ^This technique isbased

on the fact

that

healthy plants^{do not} contain detectable amounts ofdsRNA whereas CTV-infected plants ^contain

adsRNA band of13.3 x 10kd,corres- ponding to the full length replicative form of the virus plus several sub- genomic bands. The number and posi- tion of these bands are constant for each CTV isolate when assayed ⁱⁿ the same host. The current procedure for

dsRNA

analysis

consists of a phenol extraction of nueleic acids from young bark tissue, dsRNA purification by CF-11 cellulose column chromatog- raphy ^and separation ^of dsRNAs by polyacrylamide gel electrophoresis

(18, 19, 21). This is a quick and simple method for identification of CTV strains, but it hasthe following limita- tions: i) ^dsRNA

patterns

^ofsome

iso-

latesmay change depending ^onthehost and on the _season (32, 43); ii) dsRNA patterns arenot necessarily related to pathogenic capabilities of CTV ^iso- lates. Some isolates having similar biological characteristics _{may differ}in

their dsRNA profiles, whereas _some others having the same dsRNA _pat- tern may differ widely in biological be- havior (43); iii) The presence ^{of low} molecular weight dsRNA band (0.5x

10%) has been associated with severe

CTV

isolates

inducing a seedling yel- lows reaction and/or stem pitting ⁱⁿ grapefruit (19, 32). However, ⁱⁿ other citrus areas such as Spain or Corsica

14

this dsRNA band can also be found ⁱⁿ some mild reacting CTV isolates (1, 43); iv) Under

field

conditions, dsRNA recovery ^has strong seasonal vari- ations and sampling hastobe carefully timed to detect the complete dsRNA profile ^{(21, 43).}

Hybridization ^with complimen- tary ^{DNA (cDNA) probes}

to

^{the virus}

genome. This technique is based ^onthe specific hybridization of nueleic acids with complimentary strands.^The

viral

RNA

is

^usually attached to a mem- brane and the cDNA probe, labelled with Pa,, hybridizes onthe membrane whereas the unreactive probe is washed off. Reaction is detected by autoradiography ^of the membrane using a special ^film and an amplifying system. Since biological properties ^of the virus are encoded by the viral genome,

in

^theory, any variation ⁱⁿthe nucleotide sequence of the _genome should be detectable _by this system, as far as sufficiently specific cDNA probeswould be available. Inpractice, the system has not been widely exploited although there aresome indi- cations ofits increased usage.

The major limitations of this technique are:i)^Awiderange^{of highly} specificprobes^{would be}necessary^for unequivocal classification of CTV st- rains. ii) At present, the necessity of radioactive labeling of cDNA probes for sufficient sensitivityⁱⁿ the assay^is a majordrawback ofthe technique. ^A number

of

laboratories are presently tryingto developsensitive assays^with non-radioactive probes ^to transform

cDNA

hybridization

^{into an} easy ^and sensitive assay ^{similar to} ELISA.

Strain-specific ^monoclonal anti- bodies. Recently, strain discriminating antibodies have been obtained in

Florida(31, 49, 50)andⁱⁿ Taiwan (62).

These enable a quick ^and

reliable

^class-

ification of CTV isolates ⁱⁿ different serotypes by ELISA (33, 62). In Florida therehas been good correlation between CTV isolates reacting ^with monoclonal antibody MCA-13 and those inducing decline ofsweetonsour orange rootstocks(31, 50, 53, 64). This procedure ^has great potential ^for

Eleventh TOCV Conference

groupingCTV isolates and for monitor- ing movement

of

certain types^{of CTV}

inthefieldorwithin a plant, even when other non-reactive isolates are already established (53, 64). The major limita- tion of strain discriminating monoc- lonal antibodies

are:

^{i) A wide battery}

of monoclonals to different epitopes will be necessary ⁱⁿ order to get an accurate strain identification. ii) At present, ^CTV variability detected by monoclonal antibodies is only ⁱⁿ the viral coatprotein, ^whichbarely repre- sents^3%ofthegenome of CTV. Infact, pathogenic capabilities of any CTV variant may not be necessarily linked to variations

in

^thecoat protein. Inthe future, monoclonal antibodies could de-

velop to noncapsid virus-induced pro- teinsin^the

infected

plants. This would widen the possibilities for detecting genetic variability by serological methods.

Peptide map analysis ofvirion coat protein. This procedure consists of

purifying the viral coat protein, mak- ing a partial digestion with endop- roteases and separating the resulting peptides by electrophoresis (27,39).

Small changes ⁱⁿ the amino acid se- quence_in

of

^{the coat protein may result}

a differing set of peptides. Peptides can be further characterized byWest- ern blot using different monoclonal antibodies. Bythis procedure ^CTV

iso-

lates differing in biological properties and dsRNA as well as some having similar biological behavior and the same ^dsRNA

pattern

could be distin- guished ^(27). The major limitations of this procedure are: ⁱ⁾ The method is long andtediousand consequently not practical for routine identification of field strains. ii)Asⁱⁿ serological iden- tification, peptide maps only reflect variability of the coat protein which may not be related to pathogenicity.

CONCEPTS FOR PREVENTION Quarantine. In those countries where tristeza ^is not endemic, strict quarantine measures are _necessary

if

entry of CTV isolates ^is to be pre- vented. This may be a very ^difficult

Tristeza

problem. For _example, in California, movement of tristeza-infected bud- wood into the central valley has been quarantined since 1947. Yet, despite legislation and cooperation by most growers, over^25,000tristeza-infected trees were detectedⁱⁿover⁴⁰⁰proper- ties inthe central valley. Uninformed growers ^continueto bringinfected ma- terialfrom southern California, where tristeza is endemic, into the area. ^Re- cently, heavy fines were levied and severe penalties weregiven toacitrus nurseryman ^{who broke} quarantine regulations and illegally brought bud- wood into the state ^(2). This incident was primarily responsible for new legislation which now sets very ^high fines for any illegal introduction ofbud- wood into California. Unless there ^is strict enforcement plus a program for education on the importance of not bringingⁱⁿ trees or budwood into any region except via specific certification programs, quarantine^willbreakdown.

Yet, quarantine remains one of the most important means

for

^preventing

the introductionof new, exotic and po-

tentially destructive ^CTV

isolates.

In countries such as Peru, where apparentlynoquarantine program^has been enforced, importation ofsatsuma and other mandarin budwood directly fromJapanwas undoubtedly responsi- ble for introduction ofsevere ^CTV

iso-

lates resulting in the debilitation of their navel orange industry ^(59).

Eradication. Once a substantial

number

of

^{trees are} found dying due toinfection by tristeza, it may bevery difficult to prevent an epidemic. If temperature ^conditions are optimal, CTV isolates are transmissible ^and aphid vectors are abundant progres- sivelyrapiddecline may

be

inevitable.

In southern California, the fortuitous cireumstances

of

^{the early} detection of severe ^CTV

isolates

ⁱⁿthe variety^col- lection at ^UCR,

plus

^the massive infu- sion of money andenergyinto an eradi- cation program, may have prevented an epidemic. The combined efforts of the University of California and the citrus industry ^enabled the building of several greenhouses suitably staffed.

15

This made surveys, collection of bud- wood and indexing possible. This pro- gram, though considerably diminished inscope, continues on astatewidelevel (23).

The

tristeza

eradication programin the central valley of California is perhaps

the

^{oldest inthe} world for de- tection and elimination ^of

tristeza-in-

fectedtrees.Thisprogram^{began when} tristeza-infected trees were ^acciden- tally moved from southern California (where

tristeza

^{is endemic)}

to

^thecen-

tralvalley of California ^(where

tristeza

is precluded) ^(58). Initially, theinspec- tion for CTV-infected trees involved search for these accidentally intro- duced trees. However, once testing began, the number of trees found in- fected with CTV was alarming. Since its inception ^{in 1963,} over ^{4 million} trees have been indexed and tristeza

isstill beingdetected. ^This

testing

^and

eradication programⁱⁿthecentral

val-

ley continues.

It

^is primarily grower funded with direction andsupport^from the California Departmentof Food and Agriculture. Some CTV isolates ap- pearedto bespreading^more

rapidly

ⁱⁿ

certain orchards. The isolates were tested for transmissibility using A.

gossypii and were found to be 100%

transmissible (55, 56). The long term

success

of

^thismost important tristeza eradication programwill dependonthe continued support bythe growers ^and bythe California State Department ^of Agriculture. ^New ELISA _technology has help speed up detection.

The Israeli citrus tristeza eradica- tion program ^isperhaps the ^most

effi-

cient and completely computerized of any program. Bar-Joseph ^etal. (7)re- ports that between ^1970-1977, more than300,000tests were made by inde- xing to Mexican lime and by electron microscopy. Since that time many mil- lions of trees have been indexed by ELISA. However, despite the ability to index millions of trees per year ^by use of ELISA in this most efficient facility, tristeza continues to spread.

Inthe excellent and comprehensive re- viewonthecontinuedchallenge ofcon-

trol ofCTV, Bar-Joseph etal. (7)listed

16

four main factors contributing to the continuing epidemic: ^“1) allocation of limited resources ²⁾ failure to impose regulations and lack ofgrowercooper- ation in timely removal of infected trees ^{3) low} rates of CTVdetectionby ELISA incertain groves carrying^CTV isolates

that

caused a rapid CTV de- cline (9); and 4) the recent adoption of the horticultural practice of topping and hedging ^mature

citrus

^treeswhich

caused both increased aphid vector populations and acceleration ⁱⁿ the spread ^of the virus.” The reviewers further reported: “The eradication ef- forts which so far involved some six million tests atanestimated cost of US

$5 million, were not effective in sup- pressing the disease, but undoubtedly extended citrus _production onthe sour orange rootstock ^{for 5-10} years.”

In Spain, asevere ^CTV

isolate

^in-

ducing seedling yellows and stem pit- ting ⁱⁿ grapefruit ^and other citrus species was discovered ⁱⁿ anearlysat- suma cultivar illegally introduced, probablyfromJapan^{(4). Since}thisse- vere isolate was initially restricted to this particular cultivar^and

this cultivar

was new ⁱⁿ Spain, an eradication pro- gram wasestablished ⁱⁿthe Valencian Community on the following basis:

Growers who hadpropagated the early

satsuma

cultivar

had to make a manda- tory statement declaringplotsin^which theyhad made propagations. ThePlant Protection personnel took samples of these plots and indexed for CTV by ELISA.

If

^{any ofthe tests were posi-}

tive the entire plantingwas destroyed andthe growerwas compensated. Cur- rent estimates are that ^80% of propa- gated trees have been removed. Plan- tings surrounding these satsumas ^will be surveyedby ELISA for CTV

anda

percentage ^of

infected

trees ^willbe in- dexed for_presence

of

severe strainsby biological indexing or by any of the procedures described herein.

It thus appears that eradication can be effective under some circum- stances, butmay^be

difficult

^under

cer-

tain environmental conditions and

Eleventh IOCV Conference

without sufficient government ^and grower support.

Education. This remains

as

^avery

powerful and effective tool for limiting import^ofdestructive ^CTV

isolates

(or any other citrus pathogens). This is particularly true ^forareas^ofthe world (such as countries of the Mediterra- nean basin, Mexico, Central America or the Caribbean islands) where tristezais not endemic and not causing decline ofsweet_orange onsour orange rootstocks. Knowledge ofthe destruc- tive capacity and rapacity ^of tristeza has been known for many years.

It

^is estimated that over ^{50 million} trees were killed by tristeza and the virus

continues

to

^{destroyanddebilitatecit-}

rus ⁱⁿ many countries. The IOCV was founded on this need to learn about

why

tristeza

^destroysand,through the Organization and its publications, there is an excellent opportunity ^for

members

to

^{shareand use}thisinforma- tion to publicize the _many dangers ^of tristeza. Publicity mustbe repeatedly given toextension personnel, to ^local citrusjournals,

to

^{the press}and televi- sion, at airports and to travelers.

Growers, and especially nurseryman, mustbe informed and madeaware that bringing untested budwood into their citrus growing area can be potentially dangerous^and destructive to their or- chards as well as to their industry.

Educational programs should be step- ped up tomeet theeverincreasing ease of bring in budwood via modern air transport.

Itisnot

enough forinformed scientists to exchange information amongst themselves at meetings ^and through technical publications, but they have aduty to inform the _public and publicize their findings, and the findings of others. They must take leadershipand write for popularjour- nals onthe dangers

of

^{tristeza. These}

warnings should be frequent ^and con-

stantly repeated. Informed and edu- cated growers ^or nurserymen can be forearmed andthey canmaterially^aid

in not only preventing introduction of newpathogens, butⁱⁿsupporting_pro-

grams

for

education and research.

Tristeza ¹⁷

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

LITERATURE CITED

Albertini, D., R. Vogel, C. Bove andJ.M. Bove

1988. Transmission and preliminary characterization ^ofcitrustristeza^virusstrainK, p. 17-21.

In:Proc. 10th Conf. IOCV. IOCV Riverside.

Anonymous

1988. The Rio red raisesredflags. Citrograph 73(4): 77-78.

Azeri, T. and I. Karaca

1978. Investigation onthe tristeza(quick decline) virus disease inthe satsuma mandarins: its definition, crop losses and determination ofthe strainsinIzmir Province.J.^TurkishPhytopath.

7: 51-68.

Ballester-Olmos, J. F., J. A. Pina and L. Navarro

1988. Detection of a seedling yellowstristezastrainin Spain. p. 28-32. /n: Proc. 10th Conf.

TOCV. IOCV, Riverside.

Bar-Joseph, M. and G. Loebenstein

1973. Effect ofstrain, source plant, andtemperature^on transmissibility ofcitrustristeza^virus

bythemelon aphid. Phytopathology 63: 716-720.

Bar-Joseph, M.

1978. Cross protection incompleteness: ^A possible cause for natural spread ofcitrus tristeza virusaftera prolonged lagⁱⁿ Israel. Phytopathology 68: 1110-1111.

Bar-Joseph, M., R. Marcus, and R. F. Lee

1989. The continuous challenge ^ofcitrustristezavirus control. Ann. Rev Phytopathology 27:

291-316.

Bar-Joseph, M., and Y. Nitzan

1991. The spread and distribution ofcitrus tristezavirus isolates insour orange seedlings, p. 162-165. 7n: Proc. 11th Conf. IOCV. IOCV, Riverside.

Ben Ze'ev, ^1.S., M. Bar-Joseph, Y. Nitzan and Ruth Marcus

1989. Asevere^citrustristezavirus isolate causing thecollapse^oftreesof sour orange before virus is detectable throughout thecanopy. Ann. Appl. Biol. 114: 293-300.

Brlansky, R. H., R. ^R. Pelosi, S. M. Garnsey, ^{C. O.} Youtsey, R. F. Lee, R. K. Yokomi and R.

M. Sonoda

1986.Tristezaquick decline epidemicin South Florida. Proc. Fla.StateHort. Soc.99:66-69.

Calavan, E. C., M. K. Harjung, R. L. Blue, C. N. Roistacher, D.J.Gumpf, and P. W. Moore 1980.Naturalspread of seedling yellows and sweet orange andgrapefruit^stempitting tristeza virusesat the University of California, Riverside. _{p. 69-75.} /n8th Proc. Conf. IOCV. IOCV, Riverside.

Costa, A. S., andG. W. Muller

1980. Tristeza controlled by cross protection. ^A U.S.- Brazil cooperative success. Plant Dis.

64: 538-541.

Davino, M., F. Marras, F. Russo, A. Catara,^{A. Foddai andG. Terranova}

1988. Presentstatus^ofcitrustristezain Italy, p. 8-13. 7n: Proc. 10th Conf. IOCV. IOCV, Riverside.

Cox,J. E., L. R. Fraserand P. Broadbent

1976. Stempitting^ofgrapefruit: ^field protection bytheuse of mildstrains,an evaluation of trialsin two climaticdistricts,p. 68-70. /n: Proc. 7th Conf. IOCV. IOCV,Riverside.

Da Graca,

J.

^{V., L.J. Marais and L. A.} Von Broembsen

1982. Severetristeza stem pittinginyounggrapefruit. CitrusandSubtrop. FruitJ. 588: 18-21.

Da Graca,

J.

^{V., L.J.} Marais, and L. A. Von Broembsen

1984. Severetristeza^stempittingdecline of young grapefruittreesⁱⁿSouth Africa, p. 62-65.

In: Proc. 9th Conf. IOCV. IOCV, Riverside.

Dickson, R. C., M. M. Johnson, R. A. Flock, E. F. Laird, Jr.

1956. Flying aphid populations ⁱⁿ southern California citrus groves andtheir relation tothe transmission ofthetristeza virus. Phytopathology 46: 204-210.

Dodds,_1984.

J.

^A.,_Double^{R. L.}_stranded^{Jordan, J.}_RNA^A.

for

^{Heick, andthe} indexing of^{S.J. Tamaki}citrusandavocadoviruses, p. 330-336. /n:

Proc. 9th Conf. IOCV. IOCV, Riverside.

Dodds,

J.

^{A., T.Jarupat, C.}N. Roistacher, andJ.G. Lee

1987. Detection ofstrainspecific doublestranded ^RNAsin citrus species infected^withcitrus

tristezavirus: a review. Phytophylactica ^{19: 131-137.}

Dodds,

J.

^{A., R. L.Jordan, C.}N. Roistacher and T.Jarupat

1987. Diversity of citrus tristeza^virusisolates indicated by ^dsRNAanalysis. Intervirology 27: 177-188.

Dodds,

J.

^{A., T.}Jarupat, J. ^{G. Lee, and C.}N. Roistacher

1987. Effect ofstrain, host, time ofharvestand virus concentration on double-stranded RNA analysis ^ofcitrustristezavirus. Phytopathology 77: 442-447.