Biological control for grapevine crown gall using nonpathogenic Rhizobium vitis strain ARK-1

15 Pages·2017·3.46 MB·English

No.9] Proc.Jpn.Acad.,Ser.B93(2017) 755
Erratum to “Biological control for grapevine crown gall using
nonpathogenic Rhizobium vitis strain ARK-1”
[Proc. Jpn. Acad., Ser. B 93, No. 8, 547–560]
By Akira KAWAGUCHI, Koji INOUE, Koji TANINA and Mizuho NITA
In this paper, the phrases should be corrected as follows:
(page 547, line 5)
For “Communicated by Satohiko SAKAKI, M.J.A.”
Read “Communicated by Satohiko SASAKI, M.J.A.”
doi:10.2183/pjab.93.047
©2017TheJapanAcademy
No.8] Proc.Jpn.Acad.,Ser.B93(2017) 547
Review
Biological control for grapevine crown gall using
nonpathogenic Rhizobium vitis strain ARK-1
By Akira KAWAGUCHI,*1,† Koji INOUE,*2 Koji TANINA*2 and Mizuho NITA*3
(CommunicatedbySatohikoSASAKI,M.J.A.)
Abstract: Crown gall of grapevine, which is caused by tumorigenic Rhizobium vitis, is the
most important bacterial disease of grapevine throughout the world. Screening tests of biological
control agents resulted in the discovery of a nonpathogenic R. vitis strain ARK-1. By soaking
grapevine roots with a cell suspension of strain ARK-1 prior to planting in the field, ARK-1
treatmentsignificantlyreducedthenumberofplantswithcrowngallsymptoms.Severalfieldtrials
resultindicatedthatARK-1wasveryusefulinthefield,notonlyforgrapevinebutalsoforvarious
otherplantspecies.InexperimentswhereamixtureofARK-1andatumorigenicstrainata1:1cell
ratio was examined in vitro and in planta, expression levels of the virulence genes virD2 and virE2
of the tumorigenic strain were significantly lower. The suppression of virulence genes, which can
result in a reduction of gall formation and the pathogen population, seems to be a unique
mechanism of ARK-1. These results indicated that ARK-1 is a promising new agent to control
grapevine crown gall.
Keywords: Rhizobium vitis, grapevine crown gall, biological control, suppression of
virulence genes
DNA transfer from the crown gall pathogen into the
1. Introduction
plant.1) The transferred bacterial DNA is incorpo-
Crown gall disease is one of the most econom- rated into and expressed in the plant genome
ically important soil-borne bacterial plant diseases (Fig. 1), and it represents the only known example
around the world.1) Symptoms of crown gall are of DNA transfer from a bacterium to a plant in
identified as hypertrophy (increase in size) and nature.1) Other hosts of crown galls include orna-
hyperplasia (increase in the number) of plant cells, mentalwoodycropssuchasroses,Margueritedaisies,
whichresultsintheformationof“galls”ortumorson and Chrysanthemum spp., as well as vines and canes
roots and/or at the base or “crown” of woody plants such as grapevines and raspberries.1) Occasionally,
such as pome fruit (e.g., apple, pear), stone fruit field crops such as cotton, sugar beets, tomatoes,
(e.g.,cherry,apricot),andnut(e.g.,almond,walnut) beans, and alfalfa can develop crown galls, but the
trees.1) Abnormal cellular development is due to economic impact on these crops is not very high.1)
Grapevine crown gall, as one of the most
*1 Western Region Agricultural Research Center, National importantdiseasesofgrapevinearoundtheworld,2),3)
AgricultureandFoodResearchOrganization(NARO),Hiroshima, is caused mainly by Rhizobium vitis (Ti) [FAgro-
Japan. bacterium vitis (Ti), A. tumefaciens biovar 3], where
*2 Research Institute for Agriculture, Okayama Prefectural “Ti” indicates “tumor-inducing” or “tumorigenic”.
Technology Center for Agriculture, Forestry and Fisheries,
Today,R. vitis(Ti)isrecognizedasthepredominant
Okayama,Japan.
*3 AHS Jr. Agricultural Research and Extension Center, species causing grapevine crown gall. Based on
Department of Plant Pathology, Physiology, and Weed Science, reports over the past 30 years, the list of countries
VirginiaPolytechnicInstituteandStateUniversity,VA,U.S.A. with grapevine crown gall includes China, Japan,
† Correspondence should be addressed: A. Kawaguchi, SouthAfrica,Chile,Israel,manycountriesinEurope,
Western Region Agricultural Research Center, NARO, 6-12-1
the Middle East, and many countries in North and
Nishihukatsu-cho,Fukuyama,Hiroshima721-8514,Japan(e-mail:
kawaguchia240@affrc.go.jp). South America.3) Whereas in the majority of grape-
doi:10.2183/pjab.93.035
©2017TheJapanAcademy
548 A.KAWAGUCHIetal. [Vol.93,
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Fig. 1. The mechanism of crown gall disease in plant tissues. Infection of plants by Rhizobium is a multistage process. Virulent
Rhizobium strains transfer single-stranded forms of T-DNA and several virulence effector proteins through a bacterial typeIV
secretionsystemintoplanthostcells.
growing regions, freezing injuries provide sites for Virulent Rhizobium Ti strains transfer single-
initiatingcrowngalls,inSouthAfricaandIsrael,high strandformsofT-DNAandseveralvirulenceeffector
temperatures and humidity are considered equally proteinsthroughabacterialtype IVsecretionsystem
important.Crowngallscommonlyformonthetrunks into plant host cells.6),7) Pathogenicity genes of
and cordons of grapevines (Fig. 2a, b); however, Rhizobium Ti strains are mostly located on large
symptoms can even develop on 1-year-old seedlings tumor-inducing plasmids (pTi). A part of this
(Fig. 2c). Infected grapevines often produce inferior plasmid (T-DNA) is transferred and inserted into
growth, and in some cases, the disease may cause the nuclear DNA of the plant during infection.4)
partial or complete grapevine death (Fig. 2d). Subsequent expression of T-DNA genes results in
InfectionofplantsbyRhizobiumbacteria(Ti)is the overproduction of auxins and cytokinins, which
a multistage process.5) Not all steps of the infection eventually leads to abnormal gall formation in the
process for R. vitis have been identified, but known host plant. DNA genes then produce tumor-specific
processes are very similar to those of R. radiobacter compoundscalledopines,whichserveasnutrientsfor
(Ti). The first steps are chemotactic attraction R. vitis.3)
toward wounded plant cells (especially roots). Crown gall infection takes place not only in
Chemically attracted to wounds, R. vitis attaches vineyards but also in nurseries. With nursery
itself to the underground portion of the grapevine. production, symptoms develop at the site of wounds
However, in the case of grapevines, wounding events made by disbudding, at the base of rooted cuttings,
such as freezing injuries are often associated with an and at grafts; however, in many cases, the infected
outbreak of crown gall because exposed wounded plants remain symptomless until frost or other phys-
tissues aremore susceptible toinfectionbyR. vitis.3) ical damage initiates the disease.8) Since nurseries
No.8] BiologicalcontrolforgrapevinecrowngallusingnonpathogenicRhizobiumvitisstrainARK-1 549
are often located in places where the risk of frost is hybrids, and a very small production of V. labrusca)
low,infectedvinesmostlikelydonotdevelopobvious and over 260 wineries.20) After only three decades,
symptoms. Therefore, R. vitis is often transmitted the estimated annual economic income from the
through the vegetative propagation of infected Virginia wine industry has reached $1.3 billion
asymptomatic grapevines. When mother vines at a (approximately 156 billion Japanese yen) in 2015
nurseryareinfected,thepathogencanbespreadvery (newstats).WiththeEasternpartofVirginiafacing
quickly through the production and dissemination of the Atlantic Ocean and the Western part including
nursery stocks. the Appalachian Mountains, Virginia has a diverse
Themostseriousproblemswefacearethatthere rangeofclimates.21)Severewinterweather,aswellas
are no effective control methods against grapevine recent trends in extreme temperature fluctuations
crown gall. The nonpathogenic R. rhizogenes [FA. during late winter and early spring, tends to cause
rhizogenes, A. radiobacter biovar 2] strain K84 has damage to grapevine trunks, which allows the entry
been used successfully to control crown gall in many of R. vitis.3)
plant species.9),10) An agrocin produced by K84 Therefore,vinegrowersinVirginia,aswellasin
(agrocin 84), which is thought to be the primary the other northern temperate climates, including
factorinthecontrol,11)targetsatRNAsynthetasein Nagano and other northern prefectures in Japan,
tumorigenicRhizobiumstrains.12)However,K84does have been suffering from damage from crown gall
not prevent the initial infection of grapevine by on grapevines. In particular, the winters of 2013 and
R. vitis (Ti).3),8),13)–16) Although several laboratories 2014 caused significant winter injury to grapevines
have attempted to develop other biological control in Virginia and many states north of Virginia, and
agents for grapevine crown gall and reported some many growers lost a considerable amount of grape-
potential bacterial strains, they have not made a vines due to crown gall. The level of damage was
successful development until now. higher with less winter-hardy cultivars, such as
Previously, we reported that some nonpatho- MerlotandTannat.Forexample,a5-year-oldMerlot
genic R. vitis strains, which were isolated from vineyard located in the northern Virginia lost 100%
grapevine nursery stock in Japan, inhibited tumor of vines due to a combination of winter injury and
formation in some plant species including grape- crown gall (Nita, personal communication). Merlot
vine.13)–18) In this review article, we focus on non- is one of the most commonly grown wine grape
pathogenic R. vitis strain “ARK-1”, which was cultivars in Japan.22) The unpredictable weather
identified in our lab and strongly inhibited tumor patterns, lack of management strategies, and de-
formationingrapevineinfields,asanewantagonistic structive nature of this disease have made crown
strain. gall one of the most difficult diseases for northern
temperate climate viticulture.
2. The crown gall problem casts
a dark shadow over vineyards 3. Screening tests for biological control agents
Grapes are an important agricultural product 3.1. Co-inoculation of plant stems with
because they have a variety of uses, such as table pathogens/nonpathogens. Results of a field
grapes, wine production, juice production, or for survey of potential R. vitis (Ti) infections of mother
drying into raisins. In particular, there is a great plants and nursery stocks of grapevine in Okayama
demand for wine production around the world, Prefecture, Japan, revealed that there were numer-
including the recent increase in popularity in the ous nonpathogenic strains isolated on selective
Japanese wine industry. A global examples that can medium.13) Most of these strains were identified
be relevant to Japan is the State of Virginia, located as nonpathogenic R. vitis using pathogenicity tests,
in the mid-Atlantic region of the US. Although the bacteriological characteristics, specific multiplex
history of wine grape production in Virginia started polymerase chain reaction (PCR), and 16S rDNA
in 1860’s when settlers from Europe brought grape- sequencing analysis.13),16)
vines to the New World, the modern Virginia wine Screening tests for biological control agents
industry did not start until the mid-1980’s due to using nonpathogenic R. vitis by a needle-prick
many disease and insect problems.19) Currently, method were carried out,13),14),16) with a total of 306
Virginia ranks fifth in wine grape production in the nonpathogenic strains to determine their activities
US, with 1,416 hectares of wine grape production against a pathogenic strain. Cell suspensions of the
(mainly Vitis vinifera, and inter- or intra-specific pathogenicandnonpathogenic strains wereprepared
550 A.KAWAGUCHIetal. [Vol.93,
and mixed in various combinations at cell ratios of VAR03-1. After soaking, treated grapevine seedlings
1:1.Theinoculationconsistedofadropofthemixed wereplantedinpotswithsoilinfestedwithamixture
cell suspension placed into a needle-prick wound on of seven R. vitis (Ti) strains at a final concentration
thestemofatomatoorgrapevineseedling.Wefound of5#107cells/gsoilandgrowningreenhouses.14)–16)
that using tomato seedlings was appropriate in this We carried out four experiments. Tumors formed
screening test because tomato seedlings grow very on roots and a stem were studied. Treatment of
fast from seeds and tumors formed easily on the grapevinerootswithstrainsARK-1,ARK-2,ARK-3,
stemsbyinoculationwithTi(tumorigenic)strains.13) or VAR03-1 significantly reduced the percentage of
In a test with a 1:1 cell ratio of pathogen/ grapevine seedlings with tumors caused by the Ti
nonpathogen onto stems of tomato and grapevine, strains.14)–16) Treatment using ARK-1 showed the
some strains greatly suppressed tumor incidence and highest control effect and reduced the incidence of
thetumorsizeinstemscomparedtostemsinoculated galled plants by an average 85.2% of those on
with only a pathogenic strain. Then, nonpathogenic nontreated grapevine seedlings.16) Thus, we verified
R. vitisstrainsARK-1,ARK-2,ARK-3,andVAR03- that pre-inoculation of grapevine roots with ARK-1
1 were selected as biological control agents against was effective in reducing the percentage of plants
grapevine crown gall.16) with galls. These positive results encouraged us to
There are at least five genotypes in R. vitis (Ti) conductfieldtrialsontheassumptionofthepractical
based on multilocus sequencing analysis of house- agricultural condition.
keepinggenespyrG,recA,andrpoD.23)Ongrapevine
4. Field trials for practical use
seedlings, a 1:1 cell ratio of each strain ARK-1,
ARK-2,ARK-3,orVAR03-1tothemixtureofseven Field trials are an essential part of the develop-
different R. vitis (Ti) strains belonging to five differ- ment of new agricultural technology. Field trials are
ent genotypes, which were isolated from Japan, especially important in developing biological control
Australia,andGreece,significantlysuppressedtumor agents. Even though good results may be produced
incidence in stems. Among these, strain ARK-1 was in laboratory experiments, field trials often do not
foundtobethestrongestamongthestrainstestedfor show the expected results. Thus, we conducted field
inhibiting tumor formation. ARK-1 reduced tumor trials to verify the control effect of strain ARK-1 by
formation by an average of 90.6% of the positive rootdippingmethodwithnurserystocksofgrapevine
control,whichwastheoccurrenceoftumorformation in several vineyards in Japan.
after inoculating with the pathogenic strain alone From 2009 to 2013, nine field trials designed as
(Fig. 3, 4). So far, there have been no reports other randomized or systematic controlled trials of bio-
than ours that any antagonistic strains strongly logical control for grapevine crown gall were carried
inhibited tumor formation on tomato and grapevine, out at three different experimental fields (A to C) in
as ARK-1 did using the needle prick inoculation at a Okayama,Japan.Allfieldtrialsusednurserystockof
1:1 cell ratio. ARK-1 is a promising new agent to grapevine (Vitis vinifera#V. labrusca cv. Pione, or
control grapevine crown gall. V. vinifera cv. Neo Muscat). The experimental fields
Nonpathogenic R. rhizogenes strain K84, which were artificially infected with seven typical strains of
was known as a nonantagonistic strain for control R. vitis (Ti) belonging to five different genotypes.23)
grapevine crown gall, was not able to inhibit tumor Roots of plants were soaked for 1h in a cell
formation on grapevine shoots (Fig. 4).2),13)–16) In suspension of strain ARK-1 (108cells/ml) or water,
addition, a nonpathogenic R. vitis strain VAR06-30 andwerethenplantedineachplot.Thearrangement
was not ableto inhibit tumor formation (Fig. 4) and of each plot was random or systematic within each
wasnotanantagonistforgrapevinecrowngall.27)We field. Tumor formation on roots and stems of plants
are interested to clarify the mechanism of control of was investigated after 6–9 months.
ARK-1 and why ARK-1 is overwhelmingly powerful The disease incidences in the nine field trials
in suppressing tumor formation compared with K84 were subjected to meta-analysis according to a
and VAR06-30, and we discuss it in a later section. random effects model using the “DerSimonian–Laird
3.2. Biological control by a root dipping method”,becausethefieldtrialswereperformedwith
method. R. vitis (Ti) usually infects plants from different plots sizes, numbers of plots, and plants,
the soil into wounded roots. Pruned grapevine roots farms, and years. Meta-analysis is a set of statistical
were soaked for 1h in a cell suspension (108cells/ml) procedures for synthesizing research results from a
of nonpathogenic strains ARK-1, ARK-2, ARK-3, or number of different studies, and the DerSimonian–
No.8] BiologicalcontrolforgrapevinecrowngallusingnonpathogenicRhizobiumvitisstrainARK-1 551
(cid:16141) (cid:16142) (cid:16143)
(cid:16144)
Fig. 2. Thetypicalsymptomsofgrapevinecrowngall:(a)Tumorscommonlyformonthetrunksofgrapevines;(b)Tumorscommonly
formontheshootsofgrapevine;(c)Tumorsevendevelopon1-year-oldseedlings;and(d)Infestedgrapevinesoftenproduceinferior
growth,areinclinedtodropleaves,anddiseasemaycausepartialorcompletegrapevinedeath.
(cid:16141) (cid:16142)
Fig. 3. EffectofstrainARK-1ontumorformation.(a)AstemofgrapevinewasinoculatedwithmixturesoftheR.vitis(Ti)strainas
onlypathogen.Whitearrowsindicatetumorsformingattheinoculationwoundsite(b)Astemofgrapevinewasinoculatedwith
mixturesofR.vitis(Ti)strainandARK-1atthesametime,butata1:1cellratio.Whitearrowsindicatenotumorsformationon
inoculationwoundsite.Thephotographwastakenapproximately3monthsafterinoculation.
552 A.KAWAGUCHIetal. [Vol.93,
Laird method can incorporate variations among 0.001), indicating that ARK-1 treatment signifi-
studies.24) An estimate of a statistical effect, such as cantly reduced the disease incidence. The integrated
the difference in disease severity for plants with or relativeriskvalueof0.18indicatedthattheincidence
withouttreatment,iscollectedfromeachstudyalong of crown gall disease during treatment with ARK-1
with a measure of the variance of the estimate of the was decreased to 18% of that without ARK-1,
effect. The effect size of antagonist treatment was demonstrating that the control effect was very high
calculated as an integrated risk ratio. The risk ratio in the fields. Thus, the integrated relative risk value
was defined as Risk ratioF(proportion of plants of0.18indicatedthatARK-1veryusefulinthefield.
withtumorsinantagonisttreatment)/(proportionof The heterogeneity between each study was tested in
plants with tumors in water treatment). a meta-analysis of the inhibitory effects of ARK-1.
As shown in Fig. 5, the integrated relative risk The I-squared, tau-squared, and p-values were 0, 0,
was 0.18 (95% confidence interval: 0.10–0.32, p< and 0.97, respectively, indicating that heterogeneity
did not exist between field trials and that the
integrated relative risk was mainly derived from the
inhibiting effect of ARK-1. There are no reports of
(cid:16126)(cid:16090)(cid:16076)(cid:16158)(cid:16148)(cid:16149)(cid:16166)(cid:16155)(cid:16147)(cid:16145)(cid:16154)(cid:16145)(cid:16159)(cid:16159)(cid:16160)(cid:16158)(cid:16141)(cid:16149)(cid:16154)(cid:16076)(cid:16119)(cid:16100)(cid:16096)
a biological control agent that is better than ARK-1
(cid:16123)(cid:16154)(cid:16152)(cid:16165)(cid:16076)(cid:16156)(cid:16141)(cid:16160)(cid:16148)(cid:16155)(cid:16147)(cid:16145)(cid:16154)(cid:16149)(cid:16143)(cid:16076)(cid:16159)(cid:16160)(cid:16158)(cid:16141)(cid:16149)(cid:16154) (cid:16143)
for inhibiting tumor formation on the grapevines in
(cid:16126)(cid:16090)(cid:16076)(cid:16162)(cid:16149)(cid:16160)(cid:16149)(cid:16159)(cid:16076)(cid:16159)(cid:16160)(cid:16158)(cid:16141)(cid:16149)(cid:16154)(cid:16076)(cid:16130)(cid:16109)(cid:16126)(cid:16092)(cid:16098)(cid:16089)(cid:16095)(cid:16092) the field.
(cid:16126)(cid:16090)(cid:16076)(cid:16162)(cid:16149)(cid:16160)(cid:16149)(cid:16159)(cid:16076)(cid:16159)(cid:16160)(cid:16158)(cid:16141)(cid:16149)(cid:16154)(cid:16076)(cid:16109)(cid:16126)(cid:16119)(cid:16089)(cid:16095) Meta-analysis of the seven field trials showed
(cid:16126)(cid:16090)(cid:16076)(cid:16162)(cid:16149)(cid:16160)(cid:16149)(cid:16159)(cid:16076)(cid:16159)(cid:16160)(cid:16158)(cid:16141)(cid:16149)(cid:16154)(cid:16076)(cid:16109)(cid:16126)(cid:16119)(cid:16089)(cid:16094) (cid:16142) strong evidence that ARK-1 was effective in control-
(cid:16126)(cid:16090)(cid:16076)(cid:16162)(cid:16149)(cid:16160)(cid:16149)(cid:16159)(cid:16076)(cid:16159)(cid:16160)(cid:16158)(cid:16141)(cid:16149)(cid:16154)(cid:16076)(cid:16130)(cid:16109)(cid:16126)(cid:16092)(cid:16095)(cid:16089)(cid:16093) ling grapevine crown gall by application in the field.
(cid:16126)(cid:16090)(cid:16076)(cid:16162)(cid:16149)(cid:16160)(cid:16149)(cid:16159)(cid:16076)(cid:16159)(cid:16160)(cid:16158)(cid:16141)(cid:16149)(cid:16154)(cid:16076)(cid:16109)(cid:16126)(cid:16119)(cid:16089)(cid:16093) (cid:16141) Theresultsindicatedthatsoakingfor1hourinacell
suspension of 108cells/ml of ARK-1 was suitable for
(cid:16092)(cid:16076) (cid:16093)(cid:16092)(cid:16076) (cid:16094)(cid:16092)(cid:16076) (cid:16095)(cid:16092)(cid:16076) (cid:16096)(cid:16092)(cid:16076) (cid:16097)(cid:16092)(cid:16076) (cid:16098)(cid:16092)(cid:16076) (cid:16099)(cid:16092)(cid:16076) (cid:16100)(cid:16092)(cid:16076) (cid:16101)(cid:16092)(cid:16076) (cid:16093)(cid:16092)(cid:16092) practicaluse.TheeffectivenessofARKincontrolling
(cid:16128)(cid:16161)(cid:16153)(cid:16155)(cid:16158)(cid:16076)(cid:16146)(cid:16155)(cid:16158)(cid:16153)(cid:16160)(cid:16149)(cid:16155)(cid:16154)(cid:16076)(cid:16158)(cid:16141)(cid:16160)(cid:16145)(cid:16076)(cid:16084)(cid:16081)(cid:16085)
crown gall at a low cell density was advantageous
Fig. 4. Effect of co-inoculation with nonpathogenic Rhizobium in developing ARK-1 as a biopesticide. At present,
strainsandTistrainata1:1cellratioongrapevineseedlings. we verified that 5#107cells/ml of ARK-1 gave a
Dataarethemeansfromthreeexperiments.Errorbarsindicate positive effect with a relative risk value 0.15 (95%
95%confidentialintervals.Rhombilabeledwithdifferentletters
indicate a significant difference from the other bars (P<0.05, confidence interval: 0.03–0.27, p<0.05) on inhib-
Ryan’smultipletest). ition of gall formation in some field trials.
ARK-1 treatment Water treatment
Forest plot of
Plants Plants risk ratio 95%

with Total with Total confidence
Field trials tumors plants tumors plants Risk ratio interval Weight (%)
2009-A 1 30 8 30 0.13 [0.02; 0.94] 7.8%
2009-B 1 24 14 24 0.07 [0.01; 0.50] 8.4%
2010-A 0 16 4 16 0.11 [0.01; 1.90] 3.9%
2010-B 1 36 7 36 0.14 [0.02; 1.10] 7.6%
2011-A 2 20 9 20 0.22 [0.05; 0.90] 16.2%
2011-B 2 40 14 40 0.14 [0.03; 0.59] 15.9%
2012-A 4 48 14 48 0.29 [0.10; 0.81] 29.6%
2012-C 1 30 4 30 0.25 [0.03; 2.11] 7.0%
2013-A 0 38 2 40 0.21 [0.01; 4.24] 3.5%
Random effects model 282 284 0.18 [0.10; 0.32] 100%
Heterogeneity: I-squared=0%, tau-squared=0, p=0.9712
0.01 0.1 1 10 100
Fig. 5. Integratedevaluationbasedonameta-analysisoftheeffectofnonpathogenicR.vitisstrainsARK-1ongrapevinecrowngall
aftersoakingplantrootsinbacterialcellsuspensionsinfieldtrials.Thecenterandwidthofthediamondshapedemonstratethevalue
oftheintegratedrelativeriskand95%confidenceinterval,respectively.
No.8] BiologicalcontrolforgrapevinecrowngallusingnonpathogenicRhizobiumvitisstrainARK-1 553
In addition to grapevines, we have evidence On the other hand, at 12 months after inoculation
that ARK-1 can effectively control apple, Japanese with strain ARK-1sc, the bacterial population on
pear, peach, rose, and tomato crown gall caused by the root surfaces was 2#104CFU/g of the root.
R. radiobacter (Ti) and R. rhizogenes (Ti) strains, Colonization by ARK-1sc on root surfaces decreased
based on four fields and three greenhouse trials.18) to 2#102CFU/g of root after 24 months, and after
Therefore, ARK-1 effectively protects six different 30 months, ARK-1sc was not detected on root
species(grapevine,apple,Japanesepear,peach,rose, surfaces.
and tomato) of host plants against three different Thus, the result for ARK-1sc indicated that
Rhizobium Ti strain species (R. radiobacter (Ti), strain ARK-1 not only established populations in
R. rhizogenes (Ti), and R. vitis (Ti)). therhizosphere ofgrapevinebut also persistedinside
roots for up to 3 years. On the other hand, coloni-
5. Population dynamics of ARK-1
zation of ARK-1sc on root surfaces decreased to the
on roots of grapevine
detectionlimitafter30months.Thesecomparisonsof
InordertodeterminetherateofARK-1survival ARK-1sc survival inside roots and on roots surfaces
on grapevine roots in the soil, an antibiotic-resistant demonstrated that the bacterial population on root
mutant of ARK-1, ARK-1sc, was developed. ARK- surfaces was always lower than that inside the roots.
1sc was a streptomycin (St)-copper sulfate (CuSO )- ARK-1wasoriginallyisolatedfrominternaltissuesof
4
resistant mutant (St-CuSO -mutant) obtained by grapevine,13)indicatingthatARK-1isanendophytic
4
growing strain ARK-1 on a special culture medium bacterium. This phenomenon needs further inves-
withStandCuSO named“St-CuSO PotatoSucrose tigation because the ability of ARK-1 to colonize
4 4
Agar (PSA) medium”.25),26) The ARK-1sc strain was grapevine roots might affect the persistence of the
inoculatedontorootsusingtherootdippingmethod, control of crown gall by ARK-1. It is also important
and at 1-, 6-, 18-, 24-, 30-, and 36-month after to determine in which grapevine tissue ARK-1
inoculation,thecolonyformingunits(CFU)ofARK- colonizes. This can be investigated by visualizing
1scwerecountedonSt-CuSO -PSAmedia.Asshown bacterial colonization patterns with the use of a
4
in Fig. 6, 12 months after inoculation with strain luminous protein (e.g., green fluorescent protein).
ARK-1sc, the bacterial population inside roots was
2#106CFU/g (fresh weight) of the root. Coloniza- 6. Understanding the mechanism
tion by ARK-1sc inside roots remained at 5# of strain ARK-1
104CFU/gof theroot forup to24 months,andthen 6.1. Need live-cells of ARK-1. In order to
dropped to 6#103CFU/g of root after 36 months. understandthemechanismofthebiologicaleffectsof
ARK-1, an inoculation study was conducted with
three different forms of ARK-1: a cell suspension of
(cid:16093)(cid:16090)(cid:16113)(cid:16087)(cid:16092)(cid:16100)
(cid:16117)(cid:16154)(cid:16159)(cid:16149)(cid:16144)(cid:16145)(cid:16158)(cid:16155)(cid:16155)(cid:16160)(cid:16159) ARK-1 as “live cells”; a cell suspension of ARK-1
(cid:16155)(cid:16160)(cid:16155) autoclaved at 121°C in 15minutes as “dead cells”;
(cid:16158) (cid:16093)(cid:16090)(cid:16113)(cid:16087)(cid:16092)(cid:16099) (cid:16123)(cid:16154)(cid:16158)(cid:16155)(cid:16155)(cid:16160)(cid:16159)(cid:16161)(cid:16158)(cid:16146)(cid:16141)(cid:16143)(cid:16145)(cid:16159)
(cid:16091)(cid:16147) and a culture filtrate of ARK-1 by filtering with a
(cid:16129)(cid:16085) syringe filter unit (0.2µm diameter) as “culture
(cid:16114)
(cid:16084)(cid:16111) (cid:16093)(cid:16090)(cid:16113)(cid:16087)(cid:16092)(cid:16098) filtrate(CF)”.Eachtreatmentconsistedofamixture
(cid:16159)
(cid:16154)(cid:16149)(cid:16160) of ARK-1 cells and an R. vitis (Ti) (tumorigenic)
(cid:16161)(cid:16147) (cid:16093)(cid:16090)(cid:16113)(cid:16087)(cid:16092)(cid:16097) strain cells at 1:1 ratios, and these were inoculated
(cid:16154)
(cid:16153)(cid:16149) onto stems of grapevine seedlings using the needle-
(cid:16155)(cid:16158)
(cid:16146) (cid:16093)(cid:16090)(cid:16113)(cid:16087)(cid:16092)(cid:16096) prick methods described above.13),14),16)
(cid:16165)
(cid:16154)
(cid:16155) Results showed that the live strain ARK-1 in a
(cid:16155)(cid:16152)
(cid:16143) (cid:16093)(cid:16090)(cid:16113)(cid:16087)(cid:16092)(cid:16095) cell suspension significantly suppressed tumor inci-
(cid:16155)(cid:16146)
(cid:16155)(cid:16090) dence in stems compared to the control treatment
(cid:16122)
(cid:16093)(cid:16090)(cid:16113)(cid:16087)(cid:16092)(cid:16094) usingTistrains(protectivevalueF89.1%).16)Onthe
(cid:16093)(cid:16076) (cid:16098)(cid:16076) (cid:16093)(cid:16094)(cid:16076) (cid:16093)(cid:16100)(cid:16076) (cid:16094)(cid:16096)(cid:16076) (cid:16095)(cid:16092)(cid:16076) (cid:16095)(cid:16098)(cid:16076) otherhand,thedeadcellsandCFsuspensiondidnot
(cid:16121)(cid:16155)(cid:16154)(cid:16160)(cid:16148)(cid:16159)(cid:16141)(cid:16146)(cid:16160)(cid:16145)(cid:16158)(cid:16149)(cid:16154)(cid:16155)(cid:16143)(cid:16161)(cid:16160)(cid:16141)(cid:16160)(cid:16149)(cid:16155)(cid:16154) reduce tumor incidence (both protective valuesF
6.4%).16) Basically, the mechanism of antagonistic
Fig. 6. Population dynamics of nonpathogenic R.vitis strain
microorganisms as biological control agents for
ARK-1scintherootsofgrapevineafterinoculation.Dataarethe
plant disease involves bacteriocins or antibacterial
means of eight rootstocks. Error bars represent the standard
errorofthemean. materials. However, the lack of control by dead cells
554 A.KAWAGUCHIetal. [Vol.93,
and CF suspension indicated a different mechanisms and 106CFU/g in shoots without tumors.26) These
for ARK-1 and that ARK-1 may have cellular results demonstrated that ARK-1sc reduced the
functions to control crown gall formation. population of the tumorigenic bacterial population
6.2. Reducing the pathogen population at to significantly lower level by an order of ten-fold.
the wound site. A survival assay of ARK-1 was Therefore, one of the mechanisms allowing the
conducted. To differentiate the inoculated biological biologicalactivityofARK-1isasignificantinhibition
control agents from indigenous rhizobacteria, R. of a population development of the Ti strain from 7
vitis strains used in the experiment consisted of dai.
three antibiotic-resistant mutant strains, ARK-1sc, 6.3. Suppressing the expression of essential
VAR06-30sc, and VAT03-9n. ARK-1sc, as described virulencegenesinthetumorigenicstrain. Trans-
above, is an St-CuSO mutant obtained by growing formation of the host DNA by R. vitis would most
4
ARK-1 on St-CuSO -PSA medium.25),26) Nonpatho- likely to occur prior to 5 dai.28) The suppression of
4
genic R. vitis strain VAR06-30 was a nonantagon- gall formation with co-inoculation of ARK-1 and the
istic strain that was not able to inhibit tumor Ti strain occurred even with a lack of suppression
formation(Fig. 4),andaSt-CuSO mutantobtained of the Ti strain population by ARK-1 until 5 dai
4
by growing VAR06-30 on St-CuSO -PSA medium (Fig. 7a). This result indicated that the mechanism
4
was called VAR06-30sc.27) R. vitis (Ti) strain by ARK-1 toreduce crown gall symptoms might not
VAT03-9 was a tumorigenic strain isolated from involvethesuppressionoftheTistrainpopulationin
grapevine,13) and VAT03-9n was a nalidixic acid grapevine plants.
(nal)-resistant mutant (nal-mutant) obtained by The virulence (vir) genes and T-DNA are
growing on nal-PSA medium.27) The three mutants located mostly on pTi and chromosomal virulence
grew on the St-CuSO -PSA medium (ARK-1sc and genes(chv)onchromosomes.6),7)Theplantmolecules
4
VAR06-30sc) and the nal-PSA medium (VAT03-9n) acetosyringone and ,-hydroxyacetosyringone induce
at rates similar to the wild-type in PSA medium.27) the expression of the entire vir regulon in Rhizobium
Inoculation to grapevine seedlings took place with as well as the formation of T-DNA intermediate
one of three different cell suspensions: 1) ARK-1sc molecules.7) These plant molecules occur specifically
and VAT03-9n (1:1 ratio); and 2) VAR06-30sc and in the exudates from wounded and metabolically
VAT03-9n(1:1ratio).Additionalexperimentalruns active plant cells, thus, they probably allow Rhizo-
consisted of grapevine seedlings inoculated with the bium to recognize susceptible cells in nature.7) T-
each cell suspension of ARK-1sc, VAR06-30sc, or DNA transfer and processing require products of the
VAT03-9n to demonstrate the population dynamics vir genes (virA, virB, virC, virD, virE, and virG),
with single strain inoculation. which are located outside of the T-DNA coding
Populations of ARK-1sc on the plants resulted region.6),7),29),30) The expressions of virB, virC, virD,
in significantly higher number (P<0.05) than those and virE are positively regulated at the transcrip-
of VAT03-9n at 7 and 9 days after inoculation (dai) tional level by plant signal molecules.6),7),29) Two
(Fig. 7a).Ontheotherhand,populationsofVAR06- tumorigenic Rhizobium proteins, VirD2 and VirE2,
30sc and VAT03-9n did not significantly differ in directlyassociatewiththeT-DNAstrand;oneVirD2
numbers up to 9 dai (Fig. 7b). Single inoculation of molecule covalently attaches to the 5B-end of the T-
ARK-1sc,VAR06-30sc,andVAT03-9ndidnotresult DNA strand, and VirE2, a protein that binds single-
in a significant difference in population level even strandedDNA,cooperativelycoatstherestoftheT-
after 9 dai (Fig. 7c). These results suggested that DNA strand (Fig. 1).6),7),29)
ARK-1sc could not reduce the pathogen population To provide insights into that mechanism, we
at the wound site on the grapevine plants during investigated the effect of ARK-1 on suppressing the
the early period after infection, but after 7 dai, expression of two virulence genes, virD2 and virE2,
suppressed the population of VAT03-9n. The other by the Ti strain; this suppression appeared to be
result was obtained that colonization by ARK-1sc responsible for the biological control by ARK-1.
remained roughly constant at 4#107CFU/g of 6.3.1. Suppressive effect of ARK-1 co-inoculation
grapevine shoot for up to 88 dai, but colonization on the expression of the vir genes of the Ti strain. To
by Ti strain decreased to 1#106CFU g-1 of the clarify whether ARK-1 suppresses the expression
grapevine shoot after 88 dai.26) In the co-inoculation of vir genes of Ti strain VAT03-9 in planta, the
assay, the populations of Ti strain were approx- expression of virD2 and virE2 was analyzed in
imately 107CFU/g in grapevine shoots with tumors grapevine plants co-inoculated with ARK-1 and
No.8] BiologicalcontrolforgrapevinecrowngallusingnonpathogenicRhizobiumvitisstrainARK-1 555
(cid:16100) (cid:16100) (cid:16154)(cid:16159)
(cid:16086) (cid:16086)
(cid:16154)(cid:16159) (cid:16086) (cid:16142)
(cid:18436)(cid:16147)(cid:16158)(cid:16141)(cid:16156)(cid:16145)(cid:16162)(cid:16149)(cid:16154)(cid:16145)(cid:16160)(cid:16149)(cid:16159)(cid:16159)(cid:16161)(cid:16145)(cid:18437)(cid:16097)(cid:16098)(cid:16099) (cid:16141) (cid:16154)(cid:16159) (cid:16154)(cid:16159) (cid:16154)(cid:16159) (cid:16147)(cid:16145)(cid:16162)(cid:16149)(cid:16145)(cid:18437)(cid:18436)(cid:16158)(cid:16141)(cid:16156)(cid:16149)(cid:16154)(cid:16145)(cid:16160)(cid:16159)(cid:16159)(cid:16161) (cid:16097)(cid:16098)(cid:16099) (cid:16154)(cid:16159) (cid:16154)(cid:16159) (cid:16154)(cid:16159) (cid:16154)(cid:16159) (cid:16154)(cid:16159) (cid:16154)(cid:16159)
(cid:16111)(cid:16114)(cid:16129)(cid:16091)(cid:16147)(cid:16096) (cid:16147)(cid:16111)(cid:16114)(cid:16129)(cid:16091) (cid:16096) (cid:16154)(cid:16159) (cid:16130)(cid:16109)(cid:16126)(cid:16092)(cid:16098)(cid:16089)(cid:16095)(cid:16092)(cid:16159)(cid:16143)
(cid:16152)(cid:16155)(cid:16147)(cid:16092)(cid:16093)(cid:16095) (cid:16154)(cid:16159) (cid:16109)(cid:16126)(cid:16119)(cid:16089)(cid:16093)(cid:16159)(cid:16143) (cid:16152)(cid:16155)(cid:16147)(cid:16093)(cid:16092)(cid:16095) (cid:16130)(cid:16109)(cid:16128)(cid:16092)(cid:16095)(cid:16089)(cid:16101)(cid:16154)
(cid:16130)(cid:16109)(cid:16128)(cid:16092)(cid:16095)(cid:16089)(cid:16101)(cid:16154)
(cid:16094)
(cid:16094)
(cid:16092)(cid:16076) (cid:16093)(cid:16076) (cid:16094)(cid:16076) (cid:16095)(cid:16076) (cid:16097)(cid:16076) (cid:16099)(cid:16076) (cid:16101)(cid:16076) (cid:16093)(cid:16093)(cid:16076)
(cid:16092)(cid:16076) (cid:16093)(cid:16076) (cid:16094)(cid:16076) (cid:16095)(cid:16076) (cid:16097)(cid:16076) (cid:16099)(cid:16076) (cid:16101)(cid:16076) (cid:16093)(cid:16093)(cid:16076)
(cid:16112)(cid:16141)(cid:16165)(cid:16159)(cid:16141)(cid:16146)(cid:16160)(cid:16145)(cid:16158)(cid:16149)(cid:16154)(cid:16155)(cid:16143)(cid:16161)(cid:16152)(cid:16141)(cid:16160)(cid:16149)(cid:16155)(cid:16154)
(cid:16112)(cid:16141)(cid:16165)(cid:16159)(cid:16141)(cid:16146)(cid:16160)(cid:16145)(cid:16158)(cid:16149)(cid:16154)(cid:16155)(cid:16143)(cid:16161)(cid:16152)(cid:16141)(cid:16160)(cid:16149)(cid:16155)(cid:16154)
(cid:16154)(cid:16159)
(cid:16100)
(cid:16143)
(cid:18437) (cid:16154)(cid:16159)
(cid:16159)(cid:16161)(cid:16145) (cid:16099) (cid:16154)(cid:16159) (cid:16154)(cid:16159) (cid:16154)(cid:16159)
(cid:16159)
(cid:16160)(cid:16149)
(cid:16154)(cid:16145) (cid:16098) (cid:16154)(cid:16159)
(cid:16162)(cid:16149)
(cid:16145)
(cid:16141)(cid:16156) (cid:16154)(cid:16159)
(cid:18436)(cid:16147)(cid:16158) (cid:16097)
(cid:16147)
(cid:16091)
(cid:16129)
(cid:16114)
(cid:16111) (cid:16096)
(cid:16155)(cid:16147)(cid:16093)(cid:16092) (cid:16154)(cid:16159) (cid:16109)(cid:16126)(cid:16119)(cid:16089)(cid:16093)(cid:16159)(cid:16143)
(cid:16120)
(cid:16095) (cid:16130)(cid:16109)(cid:16126)(cid:16092)(cid:16098)(cid:16089)(cid:16095)(cid:16092)(cid:16159)(cid:16143)
(cid:16130)(cid:16109)(cid:16128)(cid:16092)(cid:16095)(cid:16089)(cid:16101)(cid:16154)
(cid:16094)
(cid:16092)(cid:16076) (cid:16093)(cid:16076) (cid:16094)(cid:16076) (cid:16095)(cid:16076) (cid:16097)(cid:16076) (cid:16099)(cid:16076) (cid:16101)(cid:16076) (cid:16093)(cid:16093)(cid:16076)
(cid:16112)(cid:16141)(cid:16165)(cid:16159)(cid:16141)(cid:16146)(cid:16160)(cid:16145)(cid:16158)(cid:16149)(cid:16154)(cid:16155)(cid:16143)(cid:16161)(cid:16152)(cid:16141)(cid:16160)(cid:16149)(cid:16155)(cid:16154)
Fig. 7. PopulationdynamicsofthenonpathogenicstrainsandVAT03-9nafterco-inoculationontograpevineplants.(a)Populationsof
thenonpathogenicstrainARK-1scandofthetumorigenic(Ti)strainVAT03-9nafterco-inoculationonwoundedshootsofgrapevine
seedlings at a 1:1 cell ratio. (b) Populations of the nonpathogenic strain VAR06-30sc and of the Ti strain VAT03-9n after co-
inoculationonwoundedshootsofgrapevineseedlingsata1:1cellratio.(c)PopulationsofthenonpathogenicstrainARK-1sc,of
strainVAR06-30sc,andofthetumorigenic(Ti)strainVAT03-9naftereachinoculationonwoundedshootsofgrapevineseedlings.
Dataaremeans’standarddeviationforfiveplants.(a)and(b)Significantdifferencesatagivenpointintimeareindicatedby*
(t-test,*P<0.05;**P<0.01;nsP60.05).(c)Nosignificantdifferencesatagivenpointintimeareindicatedbyns(Tukey’sHSD
test,nsP60.05).dai,daysafterinoculation.
VAT03-9. Shoots of grapevine seedlings were inocu- of ARK-1 and VAT03-9 in comparison with inocu-
latedwithnonpathogenicstrains(ARK-1orVAR06- lation with only VAT03-9 (Fig. 8). On the other
30)andTistrainVAT03-9atacellratioof1:1,and hand, virD2 and virE2 expression levels were
CF of ARK-1 plus VAT03-9 by the needle-prick significantly suppressed upon co-inoculation with
methodasdescribedabove.Shootsamplescontaining ARK-1 and VAT03-9 (Fig. 8). These results sug-
one wound site were collected, and bacterial RNA gested that treatment with ARK-1, which inhibited
was extracted. The amount of mRNA expression of tumor formation, could suppress the expression of
virD2 andvirE2oftheTistrainwereanalyzedusing virD2andvirE2bytheTistrainingrapevineplants.
areverse-transcriptionquantitativepolymerasechain On the other hand, co-inoculation with VAR06-30
reaction (RT-qPCR). and CF of ARK-1 could not suppress the expression
In plants inoculated with only VAT03-9, the of these genes, hence, and they could not inhibit
inductionofbacterialvirD2andvirE2expressionwas tumor formation.
detected at 1 dai (Fig. 8). No significant difference To evaluate the starting and ending time points
in the expression levels of virD2 and virE2 were of the suppression of vir gene expression by ARK-1,
observed upon co-inoculation with VAR06-30 or CF virD2 expression was analyzed from 1 hour after