Identification of the phytoplasma associated with peach yellows disease in northwest China

Figures & data

Fig. 1 (Colour online) Geographical distribution of the visual survey locations for peach yellows disease in Shaanxi. The symbol (+) indicates where the disease occurred and (-) for the negative locations.

Fig. 2 Symptoms in peach trees infected with phytoplasmas. The diseased peach trees exhibited yellows and dieback (A, B), small and wilted fruit (C) and tattered appearance of leaves (D).

Table 1. 16SrRNA gene sequences of phytoplasmas used for the phylogenetic analysis.

Accession no.	Associated plant disease (or host)	Strain/Acronym	16Sr group
AY389828	Aster yellows (lettuce)	AY-WB	16SrI-A
M30790	Oenothera hookeri	OAY	16SrI-B
AF222065	Clover phyllody	CPh	16SrI-C
AY265206	Paulownia witches’ broom	PaWB	16SrI-D
AY265213	Blueberry stunt	BBS3	16SrI-E
AY265211	Aster yellows (apricot)	ACLR-AY	16SrI-F
L33765	Peanut witches’ broom	PnWB	16SrII-A
U15442	Witches’ broom of lime	WBDL	16SrII-B
X83432	Faba bean phyllody	FAB	16SrII-C
Y10097	Papaya yellow crinkle	PpYC	16SrII-D
AY197656	Elm yellows (Ulmus minor)	EY-125	16SrV-A
AY197658	Elm yellows (Ulmus minor)	EY-627	16SrV-A
AY197660	Peach yellows	PY-In	16SrV-B
AB052876	Jujube witches’ broom	JWB-G1	16SrV-B
DQ919060	Jujube witches’ broom	JWB	16SrV-B
AY197659	Cherry lethal yellows (Prunus avium)	CLY5	16SrV-B
FJ572660	Apricot leaf roll	YS	16SrV-B
FJ459914	Plum roll leaf	PRL	16SrV-B
AY197654	Apocynum cannabinum	HD1	16SrV-C
AY197646	Alder yellows (periwinkle)	ALY	16SrV-C
AF305198	Parthenocissus quinquefolia	VC-F1	16SrV-C
AY197645	Flavescence dorée	FD-C	16SrV-C
AY197644	Flavescence dorée	FD-D	16SrV-D
AY197648	Rubus stunt (periwinkle)	RUS	16SrV-E
AY197649	Rubus stunt (Rubus fruticosus)	RuS400	16SrV-E
AY197650	Rubus stunt (Rubus idaeus)	RuS971	16SrV-E
AY197651	Rubus stunt (Rubus caesius)	RuSR19	16SrV-E
AY390261	Clover proliferation	CP	16SrVI-A
AF190225	‘Fragaria multicipita’	multicipita-MC	16SrVI-B
AF228053	Periwinkle little leaf	PLL-Bd	16SrVI-C
AJ542541	Apple proliferation (Malus domestica)	AP15	16SrX-A
AJ542543	Pear decline (Pyrus communis)	PD1	16SrX-C
X92869	Spartium witches’ broom	SpaWB	16SrX-D
AJ542544	European stone fruit yellows	ESFY-G1	16SrX-F
AJ964960	Grapevine yellows	2642BN	16SrXII-A
L76865	Australian grapevine yellows	AUSGY	16SrXII-B
AJ243045	Strawberry lethal yellows	SLY	16SrXII-C
AB010425	Japanese Hydrangea phyllody	JHP	16SrXII-D
DQ086423	Strawberry yellows	StrawY	16SrXII-E
M23932	Acholeplasma laidlawii strain JA1	–	–

Table 2. Results of nested PCR detection for phytoplasma in peach.

Sites	Number of samples	Number of first run-positive samples	Number of second run-positive
Baoji	10	3	4
Weinan	8	2	3
Xianyang	15	0	6
Xi’an	7	3	2
Yulin	10	0	3
Hanzhong	10	0	3

Table 3. Identities between the nine sequences determined in this study and its homogeneous sequences.

Sequences determined in this study		Sequences from NCBI				Peach yellows phytoplasma in India		Phytoplasmas of group 16SrV-B in Shaanxi		JWB -G1
Strain	Accession no.	The highest identity (%)		The lowest identity (%)		AY197660	AY332658	FJ459914	DQ919060	AB052876
PYaP-YL	KF523369	Apricot leaf roll (FJ572660)/Golden bellflower yellows (HQ844237)	99.84	Bischofia polycarpa witches’-broom (EF432569)	99.02	99.60	99.60	99.60	98.72	99.59
PYaP-WN	KF523370		99.92		99.12	99.68	99.68	99.68	98.80	99.67
PYaP-HZ	KF523371		99.84		99.00	99.60	99.60	99.60	98.72	99.59
PYaP-XY1	KF523374		99.92		99.10	99.68	99.68	99.68	98.80	99.67
PYaP-BJ1	KF523376		99.84		99.02	99.60	99.60	99.60	98.72	99.59
PYaP-XA1	KF523372	Puna chicory flat stem (JN582266)	99.84		98.94	99.52	99.52	99.68	98.64	99.51
PYaP-XY2	KF523375		99.84		98.94	99.52	99.52	99.68	98.64	99.51
PYaP-BJ2	KF523377	Jujube witches’ broom (JQ675716)	100.00		99.10	99.68	99.68	99.76	98.80	99.67
PYaP-XA2	KF523373	Apricot leaf roll (FJ572660)	100.00		99.10	99.68	99.68	99.68	98.80	99.67

Fig. 3 Transmission electron micrographs of an ultrathin section from a symptomatic sample (A) and a healthy sample (B). The black arrows indicate some phytoplasma bodies.

Fig. 4 Phylogenetic tree inferred from the 49 16S rRNA gene sequences of phytoplasmas using MEGA5. Acholeplasma laidlawii was used as an out-group reference.

Fig. 5 Separation of nested PCR products after digesting with enzymes. Lanes 1–21, PYaP. Lane 22, graft-transmitted PYaP. Lane 23, JWB. Lanes 24, CWB. Lane 25, MD. Lane M, DNA marker ΦX174 HaeIII digest DNA ladder; band sizes from top to bottom are 1353, 1078, 872, 603, 310, 281, 271, 234, 194 and 118 bp.

Fig. 6 The virtual gel image of computer-based RFLP analysis showing the distinct band profiles between peach yellows phytoplasma in Shaanxi and representatives of group 16SrV.

Fig. 7 (Colour online) The bud graft test for the peach yellows phytoplasmas in young peach trees. (A) Young peach tree just after grafting in July 2010; (B) Higher magnification of the grafting site. (C) Grafted peach tree with symptoms of yellows in July 2011.