Analysis of pathogenic and genetic variability of Corynespora cassiicola based on iPBS retrotransposons

Figures & data

Table 1. Isolates of Corynespora cassiicola used in the study.

Isolate name	Geographic origin	Host plant^a	Year^b
ZBTK1, ZBTK2	Chongqing	gynura’s deux couleur (Gynura bicolor)	2010
LJ2, LJ1	Hainan	hot pepper (Capsicum annuum)	2011
WJ1, WJ2	Shandong	lettuce (Lactuca sativa)	2010
SJD1	Hebei	bean (Phaseolus vulgaris)	2010
SC1	Shandong	lettuce (Lactuca sativa)	2011
CD2	Hebei	bean (Phaseolus vulgaris)	2009
CD3	Hainan	bean (Phaseolus vulgaris)	2010
DX	Beijing	lilac (Syzygium aromaticum)	-
KHDL	Beijing	lipstick plant (Aeschynanthus pulcher)	2007
YCH	Beijing	scarlet sage (Salvia splendens)	2007
TTN	Hainan	crane flower (Strelitzia reginae)	2010
YPH	Beijing	poinsettia (Euphorbia pulcherrima)	2006
JD1, JD4, JD2	Liaoning	cowpea (Vigna unguiculata)	2009
JD5, JD6	Hainan	cowpea (Vigna unguiculata)	2010
HCCHN05, HCCHN06, HCCHN13, HCCHN30, HCCHN42	Hainan	rubber tree (Hevea brasiliensis)	2007
HCCYN, HCCYN09, HCCYN33, HCCYN56, HCCYN61	Yunnan	rubber tree (Hevea brasiliensis)	2007
KG1	Shandong	bitter gourd (Momordica charantia)	2007
KG3	Shandong	bitter gourd (Momordica charantia)	2008
KG6	Hebei	bitter gourd (Momordica charantia)	2009
KG8	Hebei	bitter gourd (Momordica charantia)	2010
FQ1, FQ2	Beijing	tomato (Solanum lycopersicum)	2007
FQ7	Beijing	tomato (Solanum lycopersicum)	2009
FQ10	Hainan	tomato (Solanum lycopersicum)	2010
FQ13	Shandong	tomato (Solanum lycopersicum)	2011
FQ14	Zhejiang	tomato (Solanum lycopersicum)	2011
FQ16, FQ18, FQ19	Hubei	tomato (Solanum lycopersicum)	2015
QZ1	Shandong	eggplant (Solanum melongena)	2008
QZ5, QZ16	Liaoning	eggplant (Solanum melongena)	2009
QZ7, QZ8	Shandong	eggplant (Solanum melongena)	2009
QZ20	Shandong	eggplant (Solanum melongena)	2012
HB1	Hebei	cucumber (Cucumis sativus)	2008
HB22	Hebei	cucumber (Cucumis sativus)	2016
BJ6	Beijing	cucumber (Cucumis sativus)	2015
HN5, HN7	Hainan	cucumber (Cucumis sativus)	2010
LN5, LN7	Liaoning	cucumber (Cucumis sativus)	2009
ZJ2	Zhejiang	cucumber (Cucumis sativus)	2015
NM1, NM3	Neimenggu	cucumber (Cucumis sativus)	2009
YD1	Angola	cucumber (Cucumis sativus)	2010
SD1	Shandong	cucumber (Cucumis sativus)	2005
SD9	Shandong	cucumber (Cucumis sativus)	2008
SD29	Shandong	cucumber (Cucumis sativus)	2009
SD33	Shandong	cucumber (Cucumis sativus)	2015
GD2	Guangdong	cucumber (Cucumis sativus)	2014
GS1	Gansu	cucumber (Cucumis sativus)	2015
SiC1	Sichuan	cucumber (Cucumis sativus)	2015
HuB1	Hubei	cucumber (Cucumis sativus)	2015
TJ1	Tianjin	cucumber (Cucumis sativus)	2015

^aAll isolates were collected from lesions.

^b‘-’ is unknown.

Table 2. Primers used in the iPBS analyses.

iPBS primers	Sequences(5ʹ-3ʹ)	Tm (°C)	Number of bands
			Total bands	Polymorphic bands
2381^a	GTCCATCTTCCA	50	16	15
2382^a	TGTTGGCTTCCA	50	11	11
2387^a	GCGCAATACCCA	51	19	19
2389^a	ACATCCTTCCCA	50	14	12
iPBS2395^b	CCCAGCAAACCA	53	12	11

^aThe original code of each primer used has been previously listed by Kalendar et al. (2010).

^bThe original code of each primer used has been previously listed by Özer et al. (2016).

Fig. 1 A representative DNA profile of Corynespora cassiicola isolates from different hosts obtained by iPBS analysis using the iPBS primer i2395. Lanes: 1, YPH; 2, YCH; 3, SC1; 4, JD1; 5, DX; 6, KHDL; 7, TTN; 8, JD4; 9, LJ2; 10, JD2; 11, KG1; 12, KG3; 13, KG6; 14, KG8; 15, JD5; 16, JD6; 17, FQ1; 18, FQ2; 19, FQ7; 20, FQ10. M, DNA marker DL5000. The hosts of YPH, YCH, SC1, DX, KHDL, TTN and LJ2 were poinsettia (Euphorbia pulcherrima), scarlet sage (Salvia splendens), lettuce (Lactuca sativa), lilac (Syzygium aromaticum), lipstick (Aeschynanthus pulcher), crane flower (Strelitzia reginae) and hot pepper (Capsicum annuum), respectively. The host of JD1, JD2, JD4, JD5 and JD6 was cowpea (Vigna unguiculata). The host of KG1, KG3, KG6 and KG8 was bitter gourd (Momordica charantia). The host of FQ1, FQ2, FQ7 and FQ10 was tomato (Solanum lycopersicum).

Fig. 2 (Colour online) An unweighted pair group method with arithmetic mean (UPGMA) dendrogram of 69 C. cassiicola isolates obtained from Dicer’s coefficient similarity matrix using iPBS data. The vertical line is represented as a threshold line (the threshold value was 0.564). IG1 and IG2 represent two groups at the threshold value of 0.564.

Table 3. Pathogenicity profiles for 44 C. cassiicola isolates.

Isolate code	Pathogenicity^a
	Cucumber	Bitter gourd	Tomato	Eggplant	Pepper	Bean	Cowpea	Lettuce
HCCHN05	-	-	-	+	-	-	-	-
HCCHN06	-	-	-	-	-	-	-	-
HCCHN13	-	-	-	-	-	-	-	-
HCCHN30	-	-	-	+	-	-	-	-
HCCHN42	-	-	+	-	-	-	-	-
HCCYN	-	-	-	+	-	-	-	-
HCCYN09	-	-	-	-	-	-	-	-
HCCYN33	-	-	-	-	-	-	-	-
HCCYN56	-	-	-	-	-	-	-	-
HCCYN61	-	-	-	+	-	-	-	-
FQ2	-	+	+	-	-	-	+	+
FQ7	+	+	+	+	-	-	+	-
FQ10	+	+	+	+	-	+	+	-
FQ13	+	+	+	+	+	+	+	-
FQ18	+	+	+	+	-	+	+	-
FQ19	+	+	+	+	-	-	-	-
FQ16	+	+	+	+	-	-	-	-
QZ1	+	+	+	+	-	+	+	-
QZ5	+	+	+	+	-	+	-	-
QZ20	+	+	+	+	-	+	+	-
QZ8	+	+	+	-	+	-	+	-
KG3	+	+	+	+	+	+	+	-
ZBTK2	-	+	+	-	-	-	-	-
SJD1	-	-	+	+	-	+	-	-
LJ2	-	+	+	+	+	+	+	-
WJ2	+	+	+	+	-	+	+	+
SC1	+	-	+	+	-	+	+	-
CD3	+	+	+	+	-	+	+	-
JD1	-	-	+	+	-	-	+	+
DX	-	-	+	+	-	-	+	-
KHDL	-	+	+	+	-	-	+	-
YPH	-	+	+	+	-	-	+	-
YCH	+	+	-	+	-	-	-	+
TTN	-	-	-	+	-	-	-	+
NM3	+	-	+	+	-	+	-	-
SD9	+	+	+	-	+	+	+	-
SD29	+	+	+	+	-	+	+	-
YD1	+	+	+	+	-	+	+	-
LN5	+	+	+	+	-	+	+	-
HN5	+	-	+	+	-	-	+	-
HN7	+	+	+	+	-	+	+	-
TJ1	+	-	+	-	+	+	+	-
GD2	+	+	+	-	+	+	+	-
GS1	+	+	+	+	-	+	+	-

^aWhen expanding lesions in inoculated leaves were observed, the isolate was classified as pathogenic (+). When no lesion or non-expanding pinpoint lesions in inoculated leaves were observed, the isolate was classified as non-pathogenic (-).

Fig. 3 (Colour online) The unweighted pair group method with arithmetic mean (UPGMA) dendrogram of 44 C. cassiicola isolates based on the pathogenicity profiles on eight vegetable crops: cucumber, bitter gourd, tomato, eggplant, pepper, bean, cowpea and lettuce. The vertical line is represented as a threshold line (the threshold value was 0.56). PG1 and PG2 represent two groups at a threshold value of 0.56.

Table 4. Nei’s genetic identity and genetic distance of 16 populations of C. cassiicola detected by iPBS.

Population^a	CD	DX	FQ	HG	JD	KHDL	KG	LJ	QZ	SC	TTN	WJ	XJ	YCH	YPH	ZBTK
CD	****^b	0.8260	0.7267	0.8927	0.9044	0.7198	0.8595	0.6038	0.7704	0.9661	0.6908	0.6382	0.7045	0.6618	0.8260	0.7584
DX	0.1911	****	0.6948	0.8402	0.8476	0.6944	0.8078	0.5833	0.7314	0.7361	0.6667	0.6070	0.5471	0.5833	0.8333	0.6667
FQ	0.3192	0.3642	****	0.8201	0.7380	0.8846	0.9289	0.8778	0.8372	0.7134	0.8473	0.9015	0.6072	0.6677	0.6541	0.7795
HG	0.1135	0.1741	0.1983	****	0.9491	0.7851	0.9267	0.7484	0.8632	0.8709	0.6979	0.7519	0.7134	0.6872	0.8892	0.7530
JD	0.1004	0.1653	0.3039	0.0523	****	0.7144	0.9256	0.6599	0.8208	0.8628	0.6781	0.6554	0.7218	0.6418	0.9082	0.7447
KHDL	0.3288	0.3646	0.1227	0.2419	0.3363	****	0.8718	0.7500	0.7573	0.7083	0.7778	0.8023	0.5733	0.6111	0.6944	0.6944
KG	0.1514	0.2135	0.0737	0.0761	0.0773	0.1373	****	0.8637	0.8394	0.8238	0.7838	0.8597	0.6720	0.6558	0.8158	0.7758
LJ	0.5045	0.5390	0.1304	0.2898	0.4156	0.2877	0.1465	****	0.7262	0.5694	0.6389	0.9279	0.5471	0.5833	0.5833	0.6389
QZ	0.2609	0.3128	0.1777	0.1471	0.1975	0.2780	0.1751	0.3199	****	0.7418	0.8348	0.6959	0.6520	0.6901	0.7573	0.8090
SC	0.0345	0.3064	0.3377	0.1383	0.1476	0.3448	0.1939	0.5631	0.2987	****	0.6528	0.6488	0.7130.	0.6528	0.7917	0.7083
TTN	0.3699	0.4055	0.1657	0.3597	0.3885	0.2513	0.2436	0.4480	0.1806	0.4265	****	0.6628	0.5035	0.5556	0.6111	0.7778
WJ	0.4491	0.4993	0.1037	0.2851	0.4225	0.2202	0.1511	0.0748	0.3626	0.4326	0.4113	****	0.5453	0.5791	0.5791	0.6628
XJ	0.3503	0.6031	0.4990	0.3378	0.3260	0.5564	0.3975	0.6031	0.4277	0.3383	0.6863	0.6065	****	0.8585	0.6722	0.6577
YCH	0.4128	0.5390	0.4040	0.3751	0.4435	0.4925	0.4219	0.5390	0.3710	0.4265	0.5878	0.5463	0.1526	****	0.6389	0.6667
YPH	0.1911	0.1823	0.4245	0.1174	0.0963	0.3646	0.2036	0.5390	0.2780	0.2336	0.4925	0.5463	0.3971	0.4480	****	0.6667
ZBTK	0.2765	0.4055	0.2491	0.2837	0.2948	0.3646	0.2539	0.4480	0.2120	0.3448	0.2513	0.4113	0.4190	0.4055	0.4055	****

^aCD, Phaseolus vulgaris; DX, Syzygium aromaticum; FQ, Solanum lycopersicum; HG, Cucumis sativus; JD, Vigna unguiculata; KHDL, Aeschynanthus pulcher; KG, Momordica charantia; LJ, Capsicum annuum; QZ, Solanum melongena; SC, Lactuca sativa var. ramose; TTN, Strelitzia reginae; WJ, Lactuca sativa L.var. angustanaIrish; XJ, Hevea brasiliensis; YCH, Salvia splendens; YPH, Euphorbia pulcherrima; ZBTK, Gynura bicolor

^bNei’s genetic identities are presentd above the diagonal (marked by asterisks); the genetic distances are below the diagonal.

Table 5. Genetic diversity of 16 populations of C. cassiicola detected by iPBS.

Population	n^a	Pl^b	Ppl^c	Na^d	Ne^e	H^f	I^g
CD	3	11	15.28	1.1528	1.1222	0.0679	0.0972
DX	1	0	0.00	1.000	1.0000	0.0000	0.0000
FQ	9	35	48.61	1.4861	1.2645	0.1612	0.2459
HG	20	41	56.94	1.5694	1.2666	0.1721	0.2683
JD	5	27	37.50	1.3750	1.2393	0.1422	0.2116
KHDL	1	0	0.00	1.0000	1.0000	0.0000	0.0000
KG	4	32	44.44	1.4444	1.4222	0.2153	0.3008
LJ	2	0	0.00	1.0000	1.0000	0.0000	0.0000
QZ	6	30	41.67	1.4167	1.3246	0.1813	0.2608
SC	1	0	0.00	1.0000	1.0000	0.0000	0.0000
TTN	1	0	0.00	1.0000	1.0000	0.0000	0.0000
WJ	2	1	1.39	1.0139	1.0139	0.0069	0.0069
XJ	10	33	45.83	1.4583	1.1041	0.0844	0.1514
YCH	1	0	0.00	1.0000	1.0000	0.0000	0.0000
YPH	1	0	0.00	1.0000	1.0000	0.0000	0.0000
ZBTK	2	0	0.00	1.0000	1.0000	0.0000	0.0000
Average		11.67	16.20	1.1823	1.1098	0.0617	0.0966

^aSample size.

^bNumber of polymorphic loci.

^cPercentage of polymorphic loci.

^dObserved number of alleles.

^eEffective number of alleles.

^fGene diversity index.

^gShannon’s information index.

Table 6. Genetic diversity among 69 isolates of C. cassiicola based on iPBS.

	n	Na	Ne	H	I	Ht^a	Hs^b	Gst^c	Nm^d
Mean		1.9028	1.5136	0.3042	0.4598	0.3062	0.0645	0.7895	0.1333
St. Dev		0.2983	0.3401	0.1617	0.2139	0.0292	0.0019

^aTotal heterozygosity.

^bIntraspecific heterozygosity.

^cCoefficient of genetic differentiation.

^dGene flow.

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