Identification and molecular characterization of the phytoplasma associated with a lethal yellowing-type disease of coconut in Côte d’Ivoire

Figures & data

Fig. 1 Symptoms of lethal yellowing associated with the CILY phytoplasma in the Grand-Lahou region. (A) Coconut palms showing frond yellowing progressing to the younger leaves; older leaves are brown and will eventually dry up and die. (B) Coconut palms showing bare trunks corresponding to the last stage of the disease, known as ‘telephone poles’.

Fig. 2 Virtual RFLP generated from in silico digestion of the R16F2n/R2 sequences of the CILY phytoplasma and representatives from 16SrIV and 16SrXXII phytoplasma groups with AluI, BfaI, HaeIII, HpaI, HpaII and MseI. MW: phiX174DNA-HaeIII digest DNA molecular weight marker. Lane 1: CILY(Cote d’Ivoire)-16SrXXII-B (KC999037); Lane 2: CSPWD(Ghana)-16SrXXII (JQ868442); Lane 3: CSPWD(Ghana)-16SrXXII (Y13912); Lane 4: LDN(Nigeria)-16SrXXII-A (Y14175); Lane 5: LDT(Tanzania)-16SrXXII-C (FJ217385); Lane 6: LYM(Mozambique)-16SrXXII (EU549768); Lane 7: LDY(Yucatan)-16SrIV-B (U18753); Lane 8: LYJ(Jamaica)-16SrIV-A (JX560537); Lane 9: TPPD(Florida)-16SrIVD (AF434989).

Fig. 3 AluI, HaeIII, HpaI, HpaII and MseI actual RFLP patterns of the CILY phytoplasma visualized in a 3% agarose gel. Lane 1: 100 bp molecular weight marker (MM) (BioLabs, USA). Lanes 2, 3, 4, 5 and 6: AluI, HaeIII, HpaI, HpaII and MseI RFLP profiles.

Table 1. Reference strains of ‘Ca. Phytoplasma’ species and GenBank accession numbers used for RFLP and phylogenetic analyses.

Phytoplasma strain name and acronym	GenBank Accession No.	16S RFLP group
‘Ca. Phytoplasma asteris’	M30790	16SrI-B
‘Ca. Phytoplasma aurantifolia’	U15442	16SrII-B
‘Ca. Phytoplasma pruni’	JQ044393	16SrIII-A
*‘Ca. Phytoplasma palmae’	U18747	16SrIV-A
‘Lethal Decline’- Tanzania (LDT)	FJ217385	16SrIV-C
*‘Ca. Phytoplasma cocostanzaniae’	X80117	ND
‘Lethal Decline’- Tanzania (LDT)	GU952098	16SrIV-C
Texas Phoenix Palm Phytoplasma – Florida (TPPD)	AF434989	16SrIV-D
‘Lethal Decline’- Yucatan (LDY)	U18753	16SrIV-B
‘Lethal Yellowing’- Jamaica (LYJ)	JX560537	16SrIV-A
‘Ca. Phytoplasma ulmi’	AY197655	16SrV-A
‘Ca. Phytoplasma rubi’	AY197648	16SrV-E
‘Ca. Phytoplasma ziziphi’	AY072722	16SrV-B
*‘Ca. Phytoplasma vitis’	AY197645	16SrV-C
‘Ca. Phytoplasma balanitae’	AB689678	16SrV
‘Ca. Phytoplasma trifolii’	AY390261	16SrVI-A
‘Ca. Phytoplasma sudamericanum’	GU292081	16SrVI-I
‘Ca. Phytoplasma fraxini’	AF092209	16SrVII-A
*‘Ca. Phytoplasma luffae’	AF086621	16SrVIII-A
‘Ca. Phytoplasma phoenicium’	AF515636	16SrIX-D
‘Ca. Phytoplasma mali’	AJ542541	16SrX-A
‘Ca. Phytoplasma pyri’	AJ542543	16SrX-C
‘Ca. Phytoplasma spartii’	X92869	16SrX-D
‘Ca. Phytoplasma prunorum’	AJ542544	16SrX-F
‘Ca. Phytoplasma oryzae’	AB052873	16SrXI-A
*‘Ca. Phytoplasma solani’	AF248959	16SrXII-A
‘Ca. Phytoplasma australiense’	L76865	16SrXII-B
‘Ca. Phytoplasma japonicum’	AB010425	16SrXII-D
‘Ca. Phytoplasma fragariae’	DQ086423	16SrXII-E
*Mexican Periwinkle Virescence (MPV)	AF248960	16SrXIII
‘Ca. Phytoplasma cynodontis’	AJ550984	16SrXIV-A
‘Ca. Phytoplasma brasiliense’	AF147708	16SrXV-A
‘Ca. Phytoplasma graminis’	AY725228	16SrXVI-A
‘Ca. Phytoplasma caricae’	AY725234	16SrXVII-A
‘Ca. Phytoplasma americanum’	DQ174122	16SrXVIII-A
‘Ca. Phytoplasma castaneae’	AB054986	16SrXIX-A
‘Ca. Phytoplasma rhamni’	X76431	16SrXX-A
‘Ca. Phytoplasma pini’	AJ632155	16SrXXI-A
*‘Ca. Phytoplasma cocosnigeriae’-‘Lethal Decline’-Nigeria (LDN)	Y14175	16SrXXII-A
‘Côte d’Ivoire Lethal Yellowing’ (CILY)	KC999037	16SrXXII-B
‘Lethal Yellowing’- Mozambique (LYM)	EU549768	16SrXXII
‘Cape St. Paul Wilt Disease’ – Ghana (CSPWD)	JQ868442	16SrXXII
‘Cape St. Paul Wilt Disease’ – Ghana (CSPWD)	Y13912	16SrXXII
*Buckland valley grapevine yellows (BVGY)	AY083605	16SrXXIII-A
*Sorghum bunchy shoot (SBS)	AF509322	16SrXXIV-A
*Weeping tea witches’ broom (WTWB)	AF521672	16SrXXV-A
*Sugarcane phytoplasma D3T1	AJ539179	16SrXXVI-A
*Sugarcane phytoplasma D3T2	AJ539180	16SrXXVII-A
*Derbid phytoplasma (DP)	AY744945	16SrXXVIII-A
‘Ca. Phytoplasma omanense’	EF666051	16SrXXIX-A
‘Ca. Phytoplasma tamaricis’	FJ432664	16SrXXX-A
‘Ca. Phytoplasma costaricanum’	HQ225630	16SrXXXI-A
‘Ca. Phytoplasma malaysianum’	EU371934	16SrXXXII-A
‘Ca. Phytoplasma allocasuarinae’	AY135523	ND
‘Ca. Phytoplasma lycopersici’	EF199549	ND
‘Ca. Phytoplasma convolvuli’	JN833705	ND

*Incidentally cited or suggested novel species (IRPCM 2004; Wei et al. 2007).

ND: Not 16Sr RFLP group determined.

Fig. 4 Phylogenetic tree constructed using the neighbour-joining algorithm based on the R16F2n/R2 sequences of the CILY phytoplasma and reference 16Sr phytoplasma groups. ‘Ca. P. sp’: ‘Candidatus Phytoplasma sp.’ A. laidlawii: Acholeplasma laidlawii is used as the outgroup to root the tree. Phytoplasma sequences are described in Table 3.

Table 2. Similarity coefficients derived from RFLPs based on putative restriction-site analysis of nucleotide 16S rRNA gene sequences of the CILY phytoplasma (16SrXXII-B) and representative phytoplasmas of 16SrXXII and 16SrIV subgroups.

Phytoplasma 16Sr	1	2	3	4	5	6	7	8	9
1. CILY(XXII-B)	1.0	0.90	0.91	0.92	0.83	0.88	0.82	0.77	0.75
2. CSPWD(XXII)		1.0	0.95	0.99	0.91	0.98	0.87	0.85	0.85
3. CSPWD(XXII)			1.0	0.99	0.89	0.96	0.84	0.82	0.87
4. LDN(XXII-A)				1.0	0.89	0.97	0.89	0.83	0.89
5. LDT(IV-C)					1.0	0.90	0.93	0.91	0.92
6. LYM(XXII)						1.0	0.89	0.84	0.87
7. LDY(IV-B)							1.0	0.86	0.93
8. LYJ(IV-A)								1.0	0.85
9. TPPD(IV-D)									1.0

Table 3. Sequence similarities based on the 16S rDNA of the CILY phytoplasma compared with those representatives of 16SrIV and 16SrXXII phytoplasma subgroups.

	Similarity with reference phytoplasmas 16S rDNA (%)
Phytoplasma strain		1	2	3	4	5	6	7	8	9
1. CILY(XXII-B)	1	100	99.7	99.6	99.2	95.4	99.1	95.0	95.2	95.1
2.CSPWD(XXII)	2		100	99.9	99.6	93.9	99.4	93.9	94.5	95.6
3.CSPWD(XXII)	3			100	99.5	95.3	99.5	93.9	94.9	95.5
4.LDN(XXII-A)	4				100	98.9	100	94.0	94.9	95.6
5.LDT(IV-C)	5					100	92.8	96.6	96.3	96.9
6.LYM(XXII)	6						100	93.9	94.7	95.6
7.LDY(IV-B)	7							100	99.3	99.9
8.LYJ(IV-A)	8								100	99.4
9.TPPD(IV-D)	9									100

IRPCM. Phytoplasma/Spiroplasma Working Team–Phytoplasma Taxonomy Group 2004. ‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects. Int J Syst Evol Microbiol. 54:1243–1255. doi:10.1099/ijs.0.02854-0

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Wei W, Davis RE, Lee IM, Zhao Y 2007. Computer-simulated RFLP analysis of 16S rRNA genes: identification of ten new phytoplasma groups. Int J Syst Evol Microbiol. 57:1855–1867. doi:10.1099/ijs.0.65000-0

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