Integrated management of Cymbidium mosaic disease in commercial Dendrobium orchids using root endophytic fungi Piriformospora indica

Figures & data

Figure 1. Mosaic symptom on Dendrobium leaves due to Cymbidium mosaic virus.

Table 1. Treatment applied on Dendrobium orchid plants to control Cymbidium mosaic virus

Treatment number	Treatments
T1	Virus Ex (1 ml L^−1) foliar spray
T2	Acibenzolar-S-methyl (100 ppm) foliar spray
T3	Salicylic acid (150ppm) foliar spray
T4	P. indica (10^6 cfu) root inoculation
T5	P. indica (10^6 cfu) root inoculation + Virus Ex (1 ml L^−1) foliar spray
T6	P. indica (10^6 cfu) root inoculation + Salicylic acid (150ppm) foliar spray
T7	P. indica (10^6 cfu) root inoculation + Acibenzolar-S-methyl (100 ppm) foliar spray
T8	Untreated control
T9	Healthy control

Table 2. Pairwise comparison of change in virus titer of Dendrobium plants as a result of various treatments detected by CymMV-specific antibody in DAC-ELISA

Treatments	Absorbance value at 405 nm
	Before field experiment	30 DAT	After field experiment
T1-Virus Ex (1 ml L^−1) foliar spray	1.543	0.575 ^a	0.435 ^a
T2- Acibenzolar-S-methyl (100 ppm) foliar spray	1.627	0.901	0.907
T3- Salicylic acid (150ppm) foliar spray	1.514	1.797	1.764
T4- P. indica (10^6 cfu) root inoculation	1.213	0.666 ^a	0.376 ^a
T5- P. indica (10^6 cfu) root inoculation + Virus Ex (1 ml L^−1) foliar spray	1.241	0.565 ^a	0.423 ^a
T6- P. indica (10^6 cfu) root inoculation + Salicylic acid (150ppm) foliar spray	1.296	1.201	0.842
T7- P. indica (10^6 cfu) root inoculation + Acibenzolar-S-methyl (100 ppm) foliar spray	1.680	0.776 ^a	0.696
T8- Untreated control	1.822	2.378	1.735
T9- Healthy control	0.025	0.012	0.020
HSD *		0.964	1.296
SE (d)	NA	0.286	0.385
SE (m)		0.203	0.272

* Tukey’s Honestly Significant Difference (HSD).

Table 3. Pairwise comparison of Peroxidase activity (µg g−1min−1) in Dendrobium plants as a result of treatment with resistance inducing compounds, antiviral formulation and Piriformospora indica

Treatments	Activity of peroxidase (PO) (µg g^−1min^−1)
	Before treatment	1 DAT	2 DAT	5 DAT	10 DAT	20 DAT	30 DAT
T1—Virus Ex (1 ml/l) foliar spray	2.46	11.34^b	24.76^a	20.84^b	16.02^a	14.19^b	10.95^c
T2- Acibenzolar-S-methyl (100 ppm) foliar spray	2.35	10.95^b	12.76^c	8.93^d	12.03^b	10.12^c	15.48^b
T3- Salicylic acid (150ppm) foliar spray	1.94	12.10^b	10.65^c	15.48^c	8.04 ^cd	3.62^d	5.85^d
T4- P. indica (10^6 cfu) root inoculation	2.46	4.73^c	6.07^d	3.42^e	5.35^d	3.50^d	19.40^a
T5- P. indica root inoculation + Virus Ex (1 ml/l) foliar spray	2.60	11.57^b	17.35^b	19.40^b	16.79^a	8.12^c	3.62^de
T6- P. indica root inoculation + Salicylic acid (150ppm) foliar spray	2.94	4.45^c	19.68^b	26.09^a	11.13^bc	21.78^a	21.13^a
T7- P. indica root inoculation + Acibenzolar-S-methyl (100 ppm) foliar spray	2.39	19.76^a	13.13^c	21.44^b	18.10^a	13.10^b	3.50^de
T8—(Untreated control)	2.55	2.99 ^cd	3.98^de	3.61^e	4.55^de	4.07^d	5.35^d
T9- (Healthy plant)	1.45	1.08^d	1.58^e	1.73^e	1.19^e	2.17^d	2.28^e
HSD	NS	3.152	5.884	4.786	6.495	4.383	4.004
SE (d)	0.638	0.937	1.749	1.422	1.93	1.303	1.19
SE (m)	0.451	0.662	1.237	1.006	1.365	0.921	0.841

Figure 2. Transmission Electron Microscopic analysis at 80000X showing flexuous rod-shaped particles of Cymbidium mosaic virus.

Table 4. Pairwise comparison of Polyphenol oxidase activity (µg g−1min−1) in Dendrobium plants as a result of treatment with resistance inducing compounds, antiviral formulation and Piriformospora indica

Treatments	Activity of polyphenol oxidases (PPO) (µg g^−1min^−1)
	Before treatment	1 DAT	2 DAT	5 DAT	10 DAT	20 DAT	30 DAT
T1- Virus Ex (1 ml/l) foliar spray	1.10^a	2.225^a	1.768^b	2.407^ab	1.875^ab	0.333^bc	0.76 ^cd
T2- Acibenzolar-S-methyl (100 ppm) foliar spray	1.145^a	1.229^bcd	4.014^a	3.433^a	2.549^a	0.235^c	0.95 ^cd
T3- Salicylic acid (150ppm) foliar spray	1.105^a	1.978^ab	3.956^a	2.48^ab	2.995^a	0.219^c	1.05^bcd
T4- P. indica (10^6 cfu) root inoculation	0.387^c	0.372^e	0.692^c	0.775 ^cd	0.332^c	1.902^a	2.04^a
T5- P. indica root inoculation + Virus Ex (1 ml/l) foliar spray	0.435^bc	1.738^ab	1.271^bc	1.695^bc	0.951^bc	0.762^bc	1.167^abc
T6- P. indica root inoculation + Salicylic acid (150ppm) foliar spray	1.052^ab	1.272^bc	1.432^bc	1.081 ^cd	1.947^ab	0.606^bc	1.925^ab
T7—P. indica root inoculation + Acibenzolar-S-methyl (100 ppm) foliar spray	0.385^c	0.775^cde	1.790^b	1.105 ^cd	1.28^bc	1.077^ab	2.015^a
T8—(Untreated control)	0.412^bc	0.465^de	0.780^bc	0.387^d	0.445^c	0.487^bc	0.50 ^cd
T9- (Healthy plant)	0.705^abc	0.757^cde	0.830^bc	0.542^d	0.557^c	0.253^bc	0.262^d
HSD	1.064	1.295	1.716	1.685	1.888	1.368	1.451
SE (d)	0.316	0.385	1.749	0.501	0.561	0.406	0.431
SE (m)	0.224	0.272	1.237	0.345	0.397	0.287	0.305

Table 5. Pairwise comparison of Phenylalanine ammonia-lyase activity (µg g−1min−1) in Dendrobium plants as a result of treatment with resistance inducing compounds, antiviral formulation and Piriformospora indica

Treatments	Activity of phenylalanine ammonia-lyase (PAL) (µg g^−1min^−1)
	Before treatment	1 DAT	2 DAT	5 DAT	10 DAT	20 DAT	30 DAT
T1- Virus Ex (1ml/l) foliar spray	5.70	45.80^a	51.75^a	41.725^a	23.30^ab	5.125^c	4.875
T2- Acibenzolar-S-methyl (100 ppm) foliar spray	4.675	17.175^bc	25.25 ^cd	37.625^a	29.40^a	10.6^bc	4.075
T3- Salicylic acid (150ppm) foliar spray	4.675	24.575^b	33.875^bc	35.60^a	19.10^bc	9.65^bc	4.725
T4- P. indica (10^6 cfu) root inoculation	5.45	18.875^b	20.075^d	33.30^ab	13.525 ^cd	17.675^a	4.155
T5- P. indica root inoculation + Virus Ex (1ml/l) foliar spray	5.925	44.175^a	45.85^ab	39.20^a	6.30^ef	20.225^a	6.00
T6- P. indica root inoculation + Salicylic acid (150ppm) foliar spray	7.05	28.8^b	21.125 ^cd	23.775^b	14.35 ^cd	14.875^ab	4.90
T7- P. indica root inoculation + Acibenzolar-S-methyl (100 ppm) foliar spray	5.35	48.7^a	45.75^ab	31.525^ab	10.025^de	17.325^a	5.125
T8- (Untreated control)	4.175	4.375^c	4.675^e	3.475^c	3.175 ^f	4.675^c	3.85
T9- (Healthy plant)	4.75	4.575^c	3.525^e	4.875^c	4.20^ef	4.475^c	3.40
HSD	NS	22.235	22.509	18.327	10.913	10.581	NS
SE (d)	1.304	6.608	6.69	5.447	3.242	3.145	1.526
SE (m)	0.922	4.673	4.73	3.851	2.293	2.224	1.079

Figure 3. Spores of Piriformospora indica within root tissues of Dendrobium 30 days after inoculation.

Figure 4. Relative mobility (Rm) of peroxidase isoforms in Dendrobium leaves, separated by anionic polyacrylamide gel electrophoresis at 30 DAT.

Graph 1. Change in Cymbidium mosaic virus titer in Dendrobium plants before treatment and 30 days after treatment (DAT).

Graph 2. Peroxidase activity (µg g⁻¹min⁻¹) in Dendrobium plants as a result of treatment with resistance inducing compounds, antiviral formulation and Piriformospora indica.

Graph 3. Polyphenol oxidase activity (µg g⁻¹min⁻¹) in Dendrobium plants as a result of treatment with resistance inducing compounds, antiviral formulation and Piriformospora indica.

Graph 4. Phenylalanine ammonia-lyase activity (µg g⁻¹min⁻¹) in Dendrobium plants as a result of treatment with resistance inducing compounds, antiviral formulation and Piriformospora indica.