Diaporthe species associated with symptomatic and asymptomatic infection of soybean stems in Minnesota: identity, virulence, and growth characteristics

Figures & data

Fig. 1 Location (counties) in Minnesota where soybean stems were collected and tested for the presence of Diaporthe spp. (shaded). Symbols indicate where Diaporthe caulivora (●), D. longicolla (X), and D. cucurbitae (▲) were detected. Includes plants with symptoms of pod and stem blight, stem canker, and top dieback, as well as asymptomatic plants.

Table 1. Isolate code, species, geographical origin, associated symptoms, source, and virulence group for isolates of Diaporthe spp. used in these studies.

Isolate code^a	Species	Geographical origin (MN County) or state/country	Symptoms on soybean or identity of other original host	Source or Reference	Virulence group^b
Dc DA	D. caulivora	Dakota	Stem canker	This study	Moderate
Dc DB	D. caulivora	Sherburne	Stem canker	This study	High
Dc M4^a	D. caulivora	Chisago	Pod & stem blight	This study	Low
Dc M6^a	D. caulivora	Le Sueur	Pod & stem blight	This study	High
Dc M7^a	D. caulivora	Kandiyohi	Early dying	This study	High
Dc OL	D. caulivora	Olmsted	Pod & stem blight	This study	Low
Dc R6	D. caulivora	Redwood	Stem canker	This study	Moderate
Dc R10	D. caulivora	Redwood	Pod & stem blight	This study	Low
Dc R11^a	D. caulivora	Redwood	Stem canker	This study	Moderate
Dc SW	D. caulivora	Swift	Pod & stem blight	This study	Moderate
Dc W1	D. caulivora	Waseca	Pod & stem blight	This study	Low
Dl B3^a	D. longicolla	Sherburne	Stem canker	This study	High
Dl B4	D. longicolla	Sherburne	Stem canker	This study	High
Dl GO^a	D. longicolla	Goodhue	Stem canker	This study	High
Dl M1	D. longicolla	Blue Earth	Pod & stem blight	This study	High
Dl M2^a	D. longicolla	Mower	Pod & stem blight	This study	Moderate
Dl M5	D. longicolla	Jackson	Pod & stem blight	This study	High
Dl M8^a	D. longicolla	Jackson	Pod & stem blight	This study	High
Dl M9	D. longicolla	Nicollet	Pod & stem blight	This study	High
Dl M10	D. longicolla	Le Sueur	Pod & stem blight	This study	High
Dl R1	D. longicolla	Redwood	Stem canker	This study	High
Dl R2	D. longicolla	Redwood	Stem canker	This study	High
Dl W2	D. longicolla	Waseca	Pod & stem blight	This study	High
Dl W3^a	D. longicolla	Waseca	Stem canker	This study	High
CBS117168	D. aspalathi	South Africa	Aspalathus linearis	Gomes et al. 2013
CBS127268	D. caulivora	Croatia	Glycine max	Santos et al. 2011
Dc IA41^a	D. caulivora	USA, IA	G. max	Alison Robertson, IA State U.
Dcu^a	D. cucurbitae	USA, MN	G. max	This study
CBS127267	D. longicolla	Croatia	G. max	Santos et al. 2011
Dl FAU644	D. longicolla	USA, MD	G. max	Udayanga et al. 2015
Dl IA17^a	D. longicolla	USA, IA	G. max	Alison Robertson, IA State U.
Dl IA62	D. longicolla	USA, IA	G. max	Alison Robertson, IA State U.
Dp CBS 139281	D. phaseolorum	USA, DE	Phaseolus vulgaris	Udayanga et al. 2015
Ds DP0605	D. sojae	USA, MS	G. max	Udayanga et al. 2015

^aDenotes use for detailed morphological studies.

^bVirulence groups based on studies in a greenhouse: Low <46% severity; Moderate 46–71% severity; High >71% severity

Fig. 2 Phylogenetic analysis based on combined ITS and TUB DNA sequences. The tree is drawn to scale with branch lengths measured in the number of substitutions per site. Bootstrap values with 1000 replications are shown at the nodes. Isolate codes noted as DL are Diaporthe longicolla and DC are D. caulivora. The D. longicolla, D. caulivora, and D. cucurbitae sequences are from isolates collected in this study and the others are from GenBank for comparison (Table 1).

Table 2. Diaporthe spp. detected in soybean stems with different types of symptoms.

				Species identified as present^c
Stem symptom	Number of plants tested	Number from which Diaporthe was isolated^a	Number positive for Diaporthe with sPCR^b	Diaporthe longicolla	Diaporthe caulivora	Diaporthe cucurbitae
Pod & stem blight	25	14	4	8	10	0
Stem canker	46	22	5	17	9	1
Top dieback	22	1	2	2	1	0
Asymptomatic	154	N/A	73	73	0	0

^aNumber of stems from which Diaporthe spp. were isolated and later sequenced to determine species

^bNumber of stems from which isolates were not obtained but were positive for Diaporthe spp. with sPCR. These were later tested with specific quantitative PCR (qPCR) assays to determine species identification

^cIdentification based on sequencing and qPCR results

Table 3. Virulence of 11 isolates of Diaporthe caulivora (Dc) and 13 isolates of D. longicolla (Dl) on six soybean cultivars. Virulence was measured as percentage of internode that was symptomatic 6 weeks post-inoculation in a greenhouse at 24°C. Treatments in the same column not designated by the same letter are significantly different (α = 0.05).

Soybean Cultivar and Virulence (%)
Isolate	AG2107	BSR 101	Corsoy 79	IA2008R	MN1606SP	S19-A6	Average
Dc DA	59 abc	75 ab	13 bc	75 abc	55 ab	25 ef	50
Dc DB	68 abc	87 ab	64 abc	100 a	58 ab	92 abc	78
Dc M4	47 abc	25 b	3 c	0 e	14 b	0 f	15
Dc M6	86 ab	74 ab	42 abc	100 a	99 a	100 a	84
Dc M7	76 abc	100 a	23 bc	98 a	67 ab	68 bcde	72
Dc OL	72 abc	22 b	29 bc	8 e	56 ab	0 f	31
Dc R6	94 ab	75 ab	28 bc	13 e	77 ab	0 f	48
Dc R10	15 c	38 ab	31 bc	0 e	16 b	0 f	17
Dc R11	82 abc	61 ab	46 abc	0 e	88 ab	0 f	46
Dc SW	85 abc	88 ab	29 bc	8 e	88 a	0 f	50
Dc W1	36 bc	61 ab	25 bc	0 e	43 ab	0 f	27
Dc Avg.	65	64	55	37	60	26	47
Dl B3	88 ab	88 ab	74 abc	95 ab	83 ab	87 abc	85
Dl B4	68 abc	100 a	71 abc	80 bcd	98 a	87 abc	84
Dl GO	94 ab	75 ab	89 abc	82 bcd	96 a	70 bcde	84
Dl M1	96 a	87 ab	89 ab	89 abc	93 a	83 abcd	90
Dl M2	73 abc	65 ab	42 abc	74 bcd	69 ab	63 bcdef	64
Dl M5	89 ab	71 ab	100 a	92 abc	88 ab	97 ab	89
Dl M8	84 abc	98 a	100 a	79 bcd	99 a	87 abc	91
Dl M9	89 ab	86 ab	40 abc	67 cd	97 a	66 bcde	74
Dl M10	89 ab	98 a	61 abc	90 abc	87 ab	90 abc	86
Dl R1	91 ab	68 ab	46 abc	67 cd	85 ab	74 abcde	72
Dl R2	87 ab	91 ab	88 ab	48 de	83 ab	61 cde	76
Dl W2	95 ab	90 ab	81 abc	81 bcd	96 a	92 abc	89
Dl W3	88 abc	93 ab	81 abc	66 cd	87 ab	34 def	75
Dl Avg.	87	85	74	78	89	76	82
Grand Avg.	75	73	54	58	72	52

Fig. 3 Average virulence of 11 isolates of D. caulivora (cross hatch bars) and 13 isolates of Diaporthe longicolla (black bars) on six soybean cultivars. Virulence was measured as percentage of internode length with brown lesions 6 weeks after inoculation in a greenhouse at 24°C. Each cv. was analysed separately. Treatments designated by different letters are significantly different (α = 0.05).

Fig. 4 Branch dieback (average) resulting from inoculation of six soybean cultivars with 11 isolates of Diaporthe caulivora (cross hatch bars) and 13 isolates of D. longicolla (black bars), 6 weeks post inoculation in a greenhouse at 24°C. Percentage branch dieback was determined as the number of dead branches/total number of branches on each plant x 100. Each cv. was analysed separately. Treatments noted with different letters are significantly different (α = 0.05).

Fig. 5 Average colony diameter (cm) of Diaporthe caulivora (cross hatch bars) and D. longicolla (black bars) isolates after 14 d of growth on 0.5x PDA at three temperatures. Each temperature was analysed separately. Treatments designated by different letters are significantly different (α = 0.05).

Table 4. Sensitivity of 11 isolates of Diaporthe caulivora (Dc) and 13 isolates of D. longicolla (Dl) to the fungicides pyraclostrobin and tebuconazole. The EC₅₀ (µg mL⁻¹ a.i.) was determined based on radial growth on agar plates containing these fungicides. Fungicides were analysed separately. Treatments in the same column not designated by the same letter are significantly different (α = 0.05).

Isolate	Pyraclostrobin		Tebuconazole
Dc DA	0.03	abc	0.35	abcd
Dc DB	0.04	abc	0.37	abcd
Dc M4	0.11	a	0.16	bcdef
Dc M6	0.03	abc	0.34	abcd
Dc M7	0.03	bc	0.37	abcd
Dc OL	0.03	bc	0.35	abcd
Dc R6	0.05	abc	0.70	ab
Dc R10	0.08	ab	0.26	bcdef
Dc R11	0.00	c	0.32	bcd
Dc SW	0.01	c	0.26	bcdef
Dc W1	0.06	ab	0.27	abcdef
Dc Avg.	0.04	a	0.34	a
Dl B3	0.04	abc	0.22	bcdef
Dl B4	0.05	abc	0.71	ab
Dl GO	0.04	abc	0.68	abc
Dl M1	0.05	abc	0.03	g
Dl M2	0.03	bc	0.18	bcdef
Dl M5	0.03	abc	0.69	abc
Dl M8	0.03	bc	1.70	a
Dl M9	0.03	abc	0.36	bcde
Dl M10	0.03	abc	0.07	efg
Dl R1	0.06	ab	0.45	abcd
Dl R2	0.06	abc	0.16	cdef
Dl W2	0.14	ab	0.09	defg
Dl W3	0.04	abc	0.06	fg
Dl Avg.	0.05	a	0.42	a
Grand Avg.	0.05		0.38

Gomes RR, Glienke C, Videira SIR, Lombard L, Groenewald JZ, Crous PW. 2013. Diaporthe: a genus of endophytic, saprobic and plant pathogenic fungi. Persoonia - Molecular Phylogeny and Evolution of Fungi. 31(1):1–41. doi:10.3767/003158513X666844.

 PubMedGoogle Scholar

Santos JM, Vrandečić K, Ćosić J, Duvnjak T, Phillips AJL. 2011. Resolving the Diaporthe species occurring on soybean in Croatia. Persoonia - Molecular Phylogeny and Evolution of Fungi. 27(1):9–19. doi:10.3767/003158511X603719.

 PubMedGoogle Scholar

Udayanga D, Castlebury LA, Rossman AY, Chukeatirote E, Hyde KD. 2015. The Diaporthe sojae species complex: phylogenetic re-assessment of pathogens associated with soybean, cucurbits and other field crops. Fungal Biol. 119(5):383–407. doi:10.1016/j.funbio.2014.10.009.

 PubMed Web of Science ®Google Scholar