全 文 ：植物病理学报
ACTA PHYTOPATHOLOG ICA SIN ICA　36(2):109-115(2006)
Received date: 2005-02-24;Revised date: 2005-08-22
Foundation item: Th is s tudy w as supported by gran ts from the Nat ionalN aturalScience Foundation of Ch ina (30471141) and the Nat ional
‘863’ H igh-T ech Research Program (2001AA249021)
Co rrespond ing author:XU Jian-hua, professor, majo r inm olecu lar p lan t nem ato logy;E-mai l: jianhuaxu@n jau. edu. cn
Biog raphy:LONG H ai (1979- ), male, Ph. D s tudent of Nan jing Agricultural Un iversity, major in molecu lar ident ification of p lan t paras itic
nem atodes.
Development of a PCR Diagnostic for the Root-knot
NematodeMeloidogyne entero lobii
LONG Hai, LIU H ao, XU Jian-hua*
(Key Laboratory o fMon itor ing andMan agemen t o f D iseases and Insects, M in istry of A gricul ture,
Nan j ingA gricul turalUn ivers ity, N an jing 210095, Ch in a)
Abstract:The root-knot nem atodeMeloidogyne enterolobii Yang and E isenback 1983 is a poten tially mi portant
crop pathogen in Ch ina. To prov idem eans to assist in preventing the spread ofM. en terolobii, a single-step
PCR diagnosticwas developed for the nem atode. The diagnostic prim ers were designed based on an a lignm ent
of ribosom al in tergenic spacer 2 (IGS2) sequences from M. entero lobii, M. incognita , M. javanica , M.
arenaria and M. hap la. The re liability of the d iagnostic test has been valida ted by screen ing different
geographic populations of six close ly relatedMeloidogyne species and naturalm ixed soil nematode populations. The
test is fast and sensitive, it can be used for direct diagnosis of a single nematode and for detection ofM. entero lob ii
in m ixed soil nem atode popu lations.
Keywords:M eloidogyne enterolob ii;IGS2 sequence;PCR diagnostic
象耳豆根结线虫的 PCR鉴定和检测方法　龙 海 , 刘 昊 , 徐建华　(南京农业大学农业部病虫监测与治
理重点开放实验室 , 南京 210095)
摘要:象耳豆根结线虫是一种在中国具有潜在经济重要性的农作物病原物。为提供有助于控制象耳豆根结线虫传播扩散
的方法 , 研制了该线虫的快速 PCR鉴定和检测法。该方法 PCR引物的扩增目标为 rDNA-IGS2区域 , 其设计依据象耳豆根
结线虫与南方 、爪哇 、花生和北方根结线虫在该区域核酸序列的差异。通过对 6种近似根结线虫的不同地理群体及自然土
壤线虫群体的测试 , 验证了设计的 PCR引物针对象耳豆根结线虫的特异性和可靠性。本方法具有快速灵敏的特点 , 可用
于象耳豆根结线虫单条线虫的直接鉴定以及混合土壤线虫群体中象耳豆根结线虫的检测。
关键词:象耳豆根结线虫;IGS2序列;PCR鉴定和检测法
中图分类号:Q959. 172. 5;S432. 45　　　文献标识码:A　　　文章编号:0412-0914(2006)02-0109-07
　　Roo t-knot nem a todes (M eloidogyne spp. ) are
a m ajor group of pa thogens of agricultura l crops.
Spec ies ofM eloidogyne that have been recorded in
Ch ina inc lude M. incognita Chitwood 1949, M.
javan ica Ch itwood 1949 , M. arenaria Chitwood
1949,M. hap la Ch itwood 1949,M. entero lobii Yang
and E isenback 1983, M. gram inicola Golden and
B irchfield 1965 andM. kongi Y ang,W ang and Feng
1988
[ 1 - 3]
. The former four nem a tode species are dis-
tributed na tionw ide and thus econom ically importan.t
O f the less common species,M. enterolobii is increa-
singly be ing recogn ized as a potential threat to agricul-
ture in China.M. enterolobii was originally described
from infec ted pacara earpod tree (En terolobium
　
植物病理学报 36卷
con tortisiliquum ) in Ha inan Province, Ch ina[ 4] .
Recent investiga tions indicated that the nem atode has
becom e w idespread on the island
[ 5]
. Furthermore,
host range tests revealed thatM. entero lobii is able to
reproduce on a variety of econom ic crops and on re-
sistan t toma to and tobacco cultivars
[ 5]
. Thus, there is
an urgent need to takem easures to prevent the spread
ofM. enterolobii from the H ainan island to other
agricultural areas of the country.
Ava ilability of a fast and reliable d iagnostic is a
prerequisite for the effective imp lemen tation of regu-
latory m easures aim ing at preven tion of spread of
root-knot nem a todes. Currently, identification ofM.
en terolobii is m a in ly perform ed usingm orphological
characters and isozym e phenotypes. Morphological
identification dem ands considerab le sk ill and is com-
p licated by the fac t thatM. en terolobii shows fem ale
perineal patterns sim ilar to those ofM. incogn ita and
M. arenaria
[ 5]
. Isozym e phenotyp ing is re liable and
sensitive
[ 6] , but th is technique is on ly app licable to
fem ales, wh ich hinders its use in routine exam ination
of soil samp les that often con tain only second-stage
juven iles. Recen tly, a m tDNA-based PCR test has
been used to d ifferen tiateM. entero lob ii from other
m ajorM eloidogyne spp.
[ 1]
. Th is assay re lies on a
size polym orph ism in products amplified w ith two
conserved prim ers (C2F3 and MRH106) from the
in tergenic reg ion between theCOII and lrRNA genes
ofM eloidogyne spp. A problem associated w ith this
procedure is that the two prim ers are conserved
across a range of nem atode taxa
[ 7, 8]
. Therefore, the
test is inappropriate for detection ofM. enterolobii in
m ixed so il nema tode popula tions as am plification of
m any non-targe t nem atodes would reduce de tection
effic iency and com plica te result interpretation.
In the present study, we determ ined the sequence
of the ribosom al intergen ic spacer 2 (IGS2) forM.
enterolobii. W e subsequently designed species-specific
PCR prim ers forM. en terolobii by identify ing IGS2
sequence differences betweenM. en terolobii and the
four commonMeloidogyne spec iesM. incognita , M.
javanica ,M. arenaria andM. hapla. Our ob jective was
to develop a re liable and sensitive PCR assay for detec-
tion and identifica tion ofM. en terolobii.
1　M ateria ls andMethods
1. 1　Nematode popu lations
Nem atode populations used in th is study are
listed in Table 1. A ll theMeloidogyne populations
were derived from single egg-masses from field
popu lations. Species identification was based on
fem ale m ala te dehydrogenase (Mdh) and esterase
(E st) pheno types[ 6] (Table 1). M eloidogyne spp.
were routinely m a intained on tom ato cv. Jiangshu 3
at 20 - 28℃ in a greenhouse. The twom ixed nematode
popu lations (MSN1 and MSN2) used for detection
tests were isolated from natural soil sam ples from
one site infested w ithM. entero lobii and one site free
ofM. enterolobii, respectively. These m ixed populations
contained a variety of common soil nematodes inclu-
dingTylenchus sp. ,Tylenchorhynchus sp. ,D itylenchus
sp. andRhabditis sp. .
1. 2　DNA preparation
For pureM eloidogyne popula tions, egg-masses
were hand-p icked from infec ted tom ato roots and
juven iles were hatched in water at 25℃. For m ixed
nem atode popu lations, nema todes were isola ted from
approx im ately 500 g of soil bym eans of sugar flota-
tion , and further purified and concen tra ted using a
Baerm ann funne.l Tota l genom ic DNA was then
extracted from Melo idogyne juven iles or m ixed so il
nem atodes using amod ified protocol of them iniprep
m ethod of Cenis
[ 9, 10]
. To prepare DNA for PCR
amp lifica tion from a single nem atode, ind iv idual
second-stage juven iles, males or fem ales were hand-
picked and rup tured w ith a steel needle in a 5 μL
drop of lysis buffer (1×Ex Taq PCR buffer, 100μg /
mL pro teinase K;TaKaR a B iotechnology, Dalian ,
China)on a glass slide. Nem atode lysateswere then
transferred to PCR tubes and frozen for 30 m in at
110
　
　2期 LONG H ai, et a.l:Deve lopm en t of a PCR Diagnost ic for the Root-knotN ematode M eloidogyn e en terolobi i
Table1　Nematode popu lations used in th is study
Spec ies Population code O rigin
Isozym e phenotypes
M dh E st
　　PCR produc t 236 bp
M elo idogyne incognita MIFJ1 Fujian, Ch ina N1 I2 -
MIGX2 Guangx i, Ch ina N1 I2 -
M IHB1 H ubei, Ch ina N1 I2 -
MIJS3 Jiangsu, Ch ina N1 I2 -
M ISD 1 Shandong, China N1 I2 -
M ISH 1 Shangha i, China N1 I2 -
M ITW 2 Taiwan, Ch ina N1 I2 -
MIYN1 Y unnan, Ch ina N1 I2 -
MIAU1 A ustralia N1 I2 -
MIIT1 Italy N1 I2 -
M IPO 1 Por tuga l N1 I2 -
M ITH 1 Thailand N1 I2 -
M. javan ica M JFJ1 Fujian, Ch ina N1 J3 -
M JGD 1 G uangdong, China N1 J3 -
M JGX 1 Guangx i, Ch ina N1 J3 -
M JGX 2 Guangx i, Ch ina N1 J3 -
M JHN 1 H a inan, Ch ina N1 J3 -
M JYN 1 Y unnan, Ch ina N1 J3 -
M JBE1 Be lg ium N1 J3 -
M JHO 1 TheN e therlands N1 J3 -
M JHO 2 TheN e therlands N1 J3 -
M JIR1 Iran N1 J3 -
M. arenaria MA JS1 Jiangsu, Ch ina N1 A2 -
MA JS2 Jiangsu, Ch ina N1 A2 -
MA JS3 Jiangsu, Ch ina N1 A2 -
MASD1 Shandong, China N1 A2 -
MATW 1 Taiwan, Ch ina N1 A2 -
MAYN1 Y unnan, Ch ina N1 A1 -
MAHO1 TheN e therlands N1 A2 -
MAUS1 USA N1 A2 -
M. hapla MHSD1 Shandong, China H1 H1 -
MHSD2 Shandong, China H1 H1 -
MHTJ1 T ianjin, Ch ina H1 H1 -
MHYN1 Y unnan, Ch ina H1 H1 -
MHHO1 TheN e therlands H1 H1 -
MHHO2 TheN e therlands H1 H1 -
111
　
植物病理学报 36卷
Table1　Nematode popu lations used in th is study (Continued)
Spec ies Population code O rigin
Isozym e phenotypes
　Mdh E st 　　PCR produc t 236 bp
M. h ispan ica MSHN1 H a inan, Ch ina 　 N1 S2-M1 -
MSHN2 H a inan, Ch ina 　 N1 S2-M1 -
M. enterolobii MEHN 1 Danzhou, H ainan, China　 N1a VS1-S1 +
MEHN 2 Danzhou, H ainan, China　 N1a VS1-S1 +
MEHN 3 Danzhou, H ainan, China　 N1a VS1-S1 +
MEHN 4 Danzhou, H ainan, China　 N1a VS1-S1 +
MEHN 5 Q iongha i, H a inan, Ch ina　 N1a VS1-S1 +
MEHN 6 Q iongha i, H a inan, Ch ina　 N1a VS1-S1 +
MEHN 7 Q iongha i, H a inan, Ch ina　 N1a VS1-S1 +
MEHN 8 Q iongha i, H a inan, Ch ina　 N1a VS1-S1 +
MEHN 9 Chengm ai, H a inan, China　 N1a VS1-S1 +
M ixed soil nem atode
popula tion w ithM . enterolobii
MSN1 Danzhou, H ainan, China +
M ixed soil nem atode
population w ithoutM. en tero lob ii
MSN2 Danzhou, H ainan, China -
- 20℃. A fter freezing, sam ples were incubated for 1
h at 60℃ and 15m in a t95℃ before being processed
for PCR am plification.
1. 3　 IGS2 sequence characterization
The IGS2 reg ion between the 5S and 18S genes
of rDNA was amp lified fromM eloidogyne spp. using
prim ers 194 (5′-TTAACTTGCCAGATCGGACG-
3′) and 195 ( 5′-TCTAATGAGCCGTACGC-3′)
from Blok et a l.
[ 11]
. PCR was conducted in 25 μL
volum es contain ing 10 ng of templa te DNA , 0. 4
μmo l/L of each primer, 0. 2mmol /L dNTPs, 1×Ex
Taq PCR buffer (w ith 2 mmol /L MgC l2) and 0. 5
unit ofEx Taq DNA polymerase (TaKaRa B iotech-
nology). Reactionm ixtures were subjected to a pre-
heating at 94℃ for 4 m in, followed by 35 cycles of
30 s a t 94℃, 30 s at 55℃ and 1 m in at 72℃, and a
fina l incubation at 72℃ for 10m in using aMastercy-
cler Persona l therm al cycler (Eppendorf, Hamburg,
Germ any).
To ob tain the IGS2 sequence forM. en terolobii,
the PCR products am plified from populations ME-
HN1 andMEHN5 were excised from an agarose ge l
and purified using an Agarose Gel DNA Fragm ent
Recovery kit (TaKaR a B iotechnology). The ge l-
purified fragm ents were then cloned in the pGEM-T
Easy vector (Prom ega,M adison, USA). Sequencing
was done on anABI PR ISM 377-96 sequencer using
B igDye term ina tor chem istry (Perkin E lm er App lied
B iosystem s, Foster C ity, USA ). Two clones from
each popula tion were sequenced. Sequences were
comp iled using B ioEd itVersion 5 (T. A. H all, North
Carolina S tate Un iversity, USA). Sequence compari-
son of IGS2 among spec ies ofM eloidogyne was per-
form ed w ith CLUSTALW available w ith in B ioEdi.t
1. 4　Design of species-specific prmi ers and
PCR amp lification
Design of diagnostic PCR prim ers forM. ente-
rolobii was based on an alignment of IGS2 sequences
fromM. en terolobii and from M. incogn ita ,M. are-
naria ,M. javan ica andM. hapla as reported by B lok
et al.
[ 11]
andW ishartet al.
[ 12]
. F irst, a reverse prmi er
(Me-R) corresponding to a conserved sequence in
the 18S gene was designed. Then , a number of
poten tial forward prim ers spec ific forM. entero lob ii
112
　
　2期 LONG H ai, et a.l:Deve lopm en t of a PCR Diagnost ic for the Root-knotN ematode M eloidogyn e en terolobi i
were se lected w ith the a id of the software Fast PCR
(R. Ka lendar, Un iversity of Helsinki, F in land). To
determ ine a prim er pa ir that enab le reliable and
sensitive d iagnosis ofM. enterolob ii , various prim er
combinations were screened for amplification spec ific ity
and e fficiency against all populations ofMeloidogyne
spp. and m ixed soil nem atodes listed in Table 1.
D iagnostic PCR assays w ith the prim er setM e-F /R
were conducted in 12. 5 μL volum es tha t included 5
ng of template DNA , 0. 4 μmol /L of each prim er,
0. 2mmol /L dNTPs, 1×Ex Taq PCR buffer (w ith
2 mmol /L MgC l2) and 0. 5 unit ofEx Taq DNA
polym erase (TaKaRa B io technology). PCR was pro-
gramm ed as:4m in at 94℃, followed by 35 cyc les of
30 s a t 94℃, 30 s at 62℃ and 30 s a t 72℃, and a
fina l incubation for 10m in a t72℃. For am plification
from a single nem atode, 5 μL of crude nem atode
lysate was used as temp late DNA , and the PCR
program was ad justed to 40 cycles. A fter PCR , 5 μL
of am plification products were electrophoresed on a
1. 5% agarose gel, stained w ith e th idium brom ide
and v isulized under UV illum ination.
2　Resu lts
2. 1　 IGS2 sequence comparison
Amplification of the IGS2 was attemp ted on
representa tive popula tions ofM eloidogyne incognita ,
M. javanica ,M. arenaria ,M. hapla andM. enterolobii.
A m ajor fragment was am plified from each of the
popula tions tested (F ig. 1). The amp lification pro-
duc ts from the fourM. entero lobii popula tions were
sim ilar in size to each other and to those from the
other four spec ies. Sequences of the amplified frag-
ments from populationsMEHN1 andMEHN5 ofM.
en terolobii were subsequen tly de term ined. The two
fragmen ts were 781 and 782 bp in size, respective ly,
differing from each other on ly by a single nucleotide
insert ion at position 277 (-/T) (da ta not shown).
Sequence a lignm ent revea led that theM. enterolobii
IGS2 sequence was d istinct from those of the four
common root-knot nem atodes as reported by Blok et
al.
[ 11]
andW ishart et al.
[ 12] , show ing 84%, 84%,
84% and 70% identity to the orthologous sequences
ofM. incognita ,M. javanica ,M. arenaria and M.
hap la , respectively (data not shown).
　
Fig. 1　PCR products amp lified from the ribo-
som al in tergen ic spacer 2 ( IGS2 )
region from represen tative popu la-
tions ofMelo idogyne spp.
1:M . incogn ita-M IFJ1; 2:M. incognita-M IIT1;3:M.
javanica-M JGD1;4:M. javanica-M JBE1;5:M . arenaria-
MA JS1;6:M. arenaria-MAYN1;7:M . hap la-MHSD1;8:
M. hapla-MHHO1; 9:M . en terolobii-MEHN1; 10:M.
enterolobii-MEHN 2;11:M . enterolob ii-MEHN5;12:M.
enterolobii-MEHN 6;M:DL 2000 DNA m arker(TaK aRa).
2. 2　PCR diagnostic forM . entero lobii
Based on an a lignm ent of IGS2 sequences from
M. en terolobii ,M. incognita ,M. javanica ,M. arenaria
andM. hapla , a num ber of PCR primer pairs were
designed and tested for the ir am plification spec ificity
and effic iency toward M. en terolobii populations
(data not shown). Th is com prehensive analysis led
to the identification of a pair of prim ers diagnostic of
M. entero lob ii:Me-F (5′-AACTTTTGTGAAAGT-
GCCGCTG-3′) and Me-R ( 5′-TCAGTTCAG-
GCAGGATCAACC-3′). Using the prim er set
M e-F /R , a product of 236 bp was am plified from
purified DNA of theM. entero lobii popula tions and
the m ixed so il nema tode popula tion con tain ingM.
enterolobii (MSN1), no amp lifica tion was observed
from the populations ofM. incogn ita ,M. javanica ,
M. arenaria ,M. hapla and M. hispanica , and the
m ixed so il nem a tode population free ofM. entero lo-
bii (MSN2)(Fig. 2;Table 1). The sensitiv ity of the
113
　
植物病理学报 36卷
diagnostic was exam ined by amplification tests using
crude nem atode lysates. A s shown in F ig. 3, the d iag-
nostic fragment was read ily amplified from a single
second-stage juven ile, male or fema le.
　
Fig. 2　PCR products amp lified w ith prmi ers
Me-F and Me-R from purified DNA
from represen tative popu lations of
Melo idogyne spp. and from m ixed
soil nematode popu lations
1:M . incognita-M IFJ1;2:M . incogn ita-M IIT1; 3:M.
javanica-M JGD1;4:M. javanica-M JBE1;5:M . arenaria-
MA JS1;6:M. arenaria-MAYN1;7:M. hapla-MHSD1;8:
M . hapla-MHHO1;9:M. hispanica-MSHN 1; 10:M ixed
soil nem atode popu la tion w ithoutM . en tero lobii-MSN2;11:
M ixed soil nem atode population w ithM . en terolobii-MSN1;
12:M. enterolob ii-MEHN1;13:M . enterolobii-MEHN2;
14:M. enterolobii-MEHN5;M: DL 2000 DNA m arker
(TaKaRa).
　
Fig. 3　PCR products amp lified w ith prmi ers
Me-F and M e-R from sing le second-
stage juven ile ( lanes 1-8 ), fem ale
( lanes 9-12) and m ale ( lanes 13-15)
ofMelo idogyne en tero lob ii
M:DL 2000 DNA marke r(TaKaRa).
3　D iscussion
　　The utility of a m olecular d iagnostic test for
plant pathogens depends on three factors, .i e. re lia-
b ility, sensitiv ity and simp licity. F irstly, such a test
should be re liably discrim inative between the target
pa thogen and its close ly rela ted organism s, especially
those frequently found in the sam e habita.t Second ly,
it shou ld be sensitive so that low num ber of the target
pathogen can be de tected. F inally, the diagnostic proce-
dure should be simple to enab le h igh-throughpu tpro-
cessing of samp les. The PCR-based d iagnostic test
forM. en terolobii described here generally fulfils the
three requirem ents. The me thod is re liable as dif-
ferent geograph ic populations of six c losely related
M eloidogyne species and natura l m ixed soil nem a-
todes have been tested (Tab le 1). It is sim ple and
fast because only a sing le-step PCR am plification is
needed. As shown in Fig. 3 and 2, the test can be
used for direct diagnosis of a single nem atode and
for detection ofM. en terolobii in m ixed nem atode
populations. The latter feature is an apparent advantage
over the m tDNA-based PCR test[ 1] which is restricted
for detection ofM. en terolobii inm ixed soil nematode
popu lations.
In a previous report
[ 1] , we revealed that the
sequence of the intergen ic reg ion be tween the m ito-
chondrialCOII and lrRNA genes forM. entero lob ii
was iden tical to tha t forM. mayaguensis , and we
suggested tha tM. mayaguensis could be conspec ific
w ithM. enterolobii. In this study, we de term ined the
sequence of the ribosom al IGS2 region for two popu-
lations ofM. en terolobii. Th is sequence is 99% iden-
tica l to the orthologous sequence reported for one
popu lation of the nom inalM. mayaguensis
[ 11] (da ta
not shown). Th is sm a ll d ifference can be we ll in ter-
preted as sim ilar in traspec ific IGS2 sequence varia-
tion has been observed inM. javan ica
[ 11]
and M.
hap la
[ 12]
. In view of the high probability thatM.
mayaguensis is conspecific w ith M. en terolobii, the
diagnostic pr imersM e-F and Me-R have been care-
fully designed to locate to perfectly conserved se-
quence regions between the two nom inal spec ies. A l-
though not tested, the primer setM e-F /R should also
amp lify from populations form erly designated asM.
mayaguensis.
M. enterolob ii is regarded as a potentia l threat to
114
　
　2期 LONG H ai, et a.l:Deve lopm en t of a PCR Diagnost ic for the Root-knotN ematode M eloidogyn e en terolobi i
agriculture because it is highly pathogenic to a variety
of econom ic crops and to resistant tobacco and tomato
cultivars
[ 5]
. Considering the fact that the nem atode
has so far on ly been detected from Hainan island in
Ch ina
[ 1, 4, 5] , it is h igh ly desirable tha t regulatory
m easures should be taken to prevent the dispersal of
the pest into other agricultural areas in the country.
A s discussed above, the molecular diagnostic test pre-
sen ted here has severa l advan tages over conventional
diagnostic procedures based on m orpho logy and
isozyme pheno types. It can be exp lo ited as a conve-
n ient tool for routine de tec tion and iden tifica tion of
M. enterolobii in agricu ltural m aterials from the
Ha inan island, and should con tribute to reduc ing the
econom ic threat ofM. en terolobii.
References
[ 1] 　X u JH , L iu P L, M eng Q P, et a l. Character iza tion of
M elo idogyne spec ies from Ch ina us ing isozym e pheno-
types and amplified m itochondr ial DNA restr ic tion
fragment leng th polymorphism [ J] . European Journa l
of P lant Pa tho logy, 2004, 110:309 -315.
[ 2] 　Zhao H H, L iu W Z, L iang C, et al. M eloidogyne
gram inico la , a new record spec ies from Ch ina ( in
Ch inese) [ J] . Ac ta Phytopatholog ica S in ica(植物病
理学报), 2001, 31(2):184 -188.
[ 3] 　Y ang B J,W ang Q L, Feng R Z.M eloidogyne kongi n.
sp. (Nema toda:Melo idogynidae), a root-kno t nem a-
tode paras itiz ing Citrus sp. in G uangxi, Ch ina ( in
Ch inese) [ J] . Journa l ofG uangx iA gricu ltura lCollege
(广西农学院学报), 1988, 7(3):1 - 9.
[ 4] 　Y ang B J, E isenback J D. M eloidogyne en terolobii n.
sp. (M eloidogynidae), a root-knot nema tode parasiti-
zing pacara earpod tree in Ch ina [ J] . Journal ofN em a-
tology, 1983, 15:381 -391.
[ 5] 　L iu H, Long H , Y anX N , et al. Iden tif ica tion and host
range testing of a root-kno t nem atode infec ting guava
in H a inan P rovince( in Chinese) [ J] . Journa l of Nan-
jing Agr icultural University(南京农业大学学报 ),
2005, 28(4):55 -59.
[ 6] 　Esbenshade P R, Triantaphyllou A C. Use of enzyme
phenotypes for iden tif ica tion ofM elo idogyne spec ies
[ J] . Journal of Nema tology, 1985, 17:6 - 20.
[ 7] 　Powers T O, H arr is T S. A polym erase cha in reac tion
m ethod for iden tif ica tion of five ma jorM eloidogyne
species [ J] . Journa l ofN em atology, 1993, 25:1 -6.
[ 8] 　S tanton J, H uga ll A, Mor itz C. N uc leotide polymor-
phisms and an im proved PCR-based m tDNA diagnostic
for parthenogene tic roo t-knot nem atodes(M eloidogyne
spp. ) [ J] . Fundamenta l and Applied Nema to logy,
1997, 20:261 - 268.
[ 9] 　Cen is J L. Identification of four m ajorM eloidogyne
spp. by random amplified po lymorphic DNA (RAPD-
PCR) [ J] . Phytopa thology, 1993, 83:76 - 80.
[ 10] 　X u J H , N arabu T, M izukubo T, et a l. A mo lecular
m arker correla ted w ith se lected virulence against the
tom ato res istance geneM i inM elo idogyne incognita ,
M. javanica and M . arenaria [ J] . Phytopatho logy,
2001, 91:377 - 382.
[ 11] 　Blok V C, Phillips M S, Farge tte M. Comparison of
sequences from the ribosoma lDNA intergenic reg ion of
M eloidogyne mayaguensis and other m a jor tropical
root-knot nem atodes [ J] . Journal of Nema to logy,
1997, 29:16 - 22.
[ 12] 　W ishar t J, Ph illipsM S, Blok V C. R ibosom al interge-
n ic spacer:a polymerase chain reaction d iagnostic for
M eloidogyne chitwoodi, M. fallax , andM. hapla [ J] .
Phytopathology, 2002, 92:884 - 892.
责任编辑:　于金枝
115