Variation of Resistance and Physiological Response of Orchid from Induction Trichoderma to Infection Odontoglossum ringspot virus (ORSV)

MATERIALS AND METHODS

Research area and collection of sample: This research was carried out at the greenhouse and Laboratory of Botany, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Lampung, from January to May, 2023.

Research materials include plantlets of orchids (P. amabilis and D. discolor) 12 months old from Anggrek Bandung (West Java, Indonesia), potato dextrose agar (PDA) powder (Merck), carborundum, phosphate buffer, alcohol, aquadest, petri dish, mortar, pestle, tweezers, measuring glass, erlenmeyer, micropipette, filter paper, erlenmeyer, test tube, balance, magnetic stirrer, hot plate, volumetric flask, vortex mixer (Sigma Aldrich, USA), centrifuge (Hettich, Germany), UV-Vis spectrophotometer (Hitachi, Japan), autoclave (Memmert, Germany) and freezer (Modena, Italia).

Methodology: The study was arranged for 2 treatment factors and 4 replications with a factorial block randomized design. Orchid type (factor 1): P. amabilis (A1) and D. discolor (A2). Inoculation treatment (factor 2): Control (K), mycorrhiza (M), virus (V) and mycorrhizal-virus (MV). After 8 months of age, the orchid plantlets were acclimatized. Orchid care routinely in the greenhouse until 3-4 leaves appear. The OMF was grown on a PDA medium in a Petri dish. The OMF isolates were subcultured on media and incubated for 7 days at room temperature. The orchids were then placed in a Petri dish containing OMF and incubated for 72 hrs. Furthermore, the orchid was replanted in a sterile moss growing medium.

Inoculation of ORSV was carried out using inoculum resulting from virus multiplication on ORSV-infected tobacco plants^8,13. Tobacco leaves are weighed 1 g and then crushed in cold conditions with a mortar and pestle that has been sterilized using 70% alcohol. The mashed leaves are then added with phosphate buffer on a ratio of 1:10 (m/v). This ORSV culture was then poured onto the leaves with carborundum on the top surface¹. Inoculation was done slowly in the direction of the leaf reinforcement using a cotton bud and waiting until it dries. Furthermore, the inoculated leaves were washed in running water to remove carborundum on the leaf surface. Observations are carried out periodically until the plants show symptoms including yellowing, chlorosis, necrosis, mosaic, streak, curling and leaf malformations^3,5,8,11,13.

Analysis of disease symptom: This analysis was carried out by observing variations in symptoms of infection and incubation time on leaves that had been inoculated with the virus and OMF. The results of subsequent observations were carried out by matching the documentation of research results with reported literature^8,11-13.

Analysis of disease progression: The stages of this research were carried out by observing the incidence of the disease and the severity of the disease to determine the progress of the disease. Disease incidence was obtained by calculating the number of infected plant organs from all plant samples. While the severity of the disease is the proportion of infected plants to the total surface area of the plants observed. The intensity of the disease was obtained by calculating the damage scale (%) of the disease that appeared on the host plant. Observation of the severity of the disease is done visually. Calculation of the percentage of infected plants is carried out if the disease-causing infection occurs systematically so that the internal symptoms may have spread to all parts of the leaf even though the external symptoms are only slightly visible. The calculation of disease severity score categories was shown in Table 1^12,13.

Analysis of the level of resistance of orchids to disease infection was carried out according to the method of Mahfut et al.⁸ as shown in Table 2.

Analysis of chlorophyll: The orchid leaves that had been taken from the midrib as a result of the inoculation were weighed to a maximum of 0.01 g, added to 10 mL of ethanol and then ground with a mortar. Whatman No. 1 paper was used to filter the solution before it was added to the flacon.

The 1 mL of the sample and 1 mL of the standard solution were added to several cuvettes. With three repeats, absorption measurements were taken with a UV spectrophotometer at wavelengths of 648 and 664 nm. Chlorophyll content was calculated using UV-Vis spectrophotometer (Hitachi, Japan) with the following procedure⁸:

Statistical analysis: Data analysis adopted the statistical method of Mahfut et al.^8,13 using Analysis of Variance (ANOVA) with a significance level of 5%. The data was previously homogenized using the Levene Test. Then it was analyzed using ANOVA and continued with the Tukey’s Test at the 5% level.

RESULTS

Symptoms of disease: The results of mycorrhizal induction and ORSV inoculation on P. amabilis showed a variety of infection symptoms and different incubation times. Some of the symptoms were mild mosaic and necrotic in the treatment of orchids inoculated with viruses and mycorrhizae in replicate 1 (MA1VU1) and replicate 4 (MA1VU4) on 10th day after treatment. In addition, there was clear mosaic in the treatment MA1VU3, as well as mild mosaic and necrotic in MA1VU4 at 13th day, respectively. Other symptoms were clear mosaic in MA1VU3, as well as clear and necrotic mosaic on MA1VU2 at 18th day, respectively. Furthermore, the symptoms of MA1VU1 developed into necrosis on 15th day.

Whereas, in D. discolor, the results showed mosaic in the MA2VU1 and MA2VU3 treatments on the 10th day, clear mosaic on MA2VU2 on the 13th days, mosaic on MA2VU3 and MA2VU4 on the 18th day. In MA2VU2 there is a mixture of symptoms, namely mild mosaic and necrotic. In addition, the MA2VU1 treatment was symptomless until the end of the observation. Variations of symptoms of infection and incubation period resulting from mycorrhizal induction and ORSV inoculation in P. amabilis and D. discolor were shown in Table 3.

Symptoms in P. amabilis indicate the development of infection, namely MA1VU1, the color of the leaves changes from green to brown and necrosis appears on the edges of leaves A (V1) and leaves B (V2). In MAVU2 there is necrotic on leaf C (V1), also necrotic and mosaic on leaf D (V2). In MA1VU3 there is a mosaic from the middle to the tip of the E (V1) leaf, while in the F (V2) leaf, there is a mosaic on the entire leaf blade.

On MA1VU4 there was a mild mosaic on the entire surface of the leaf and necrotic in the middle of the G (V1) leaf, while on the H (V2) leaf there were symptoms of a mild and necrotic mosaic.

Whereas, in D. discolor, MA2V1 there was also a change in symptoms to a mosaic in the middle of the leaf on leaf A (V1) (Fig. 1a), while on leaf B (V2) it showed symptomlessly (Fig. 1b). In MAVU2 there is a mosaic from the base to the middle of the leaf and there are brown necrotic spots on the leaf edges on leaves C (V1) and D (V2) Fig. 1(c-d). In MA2VU3 there were mild mosaic changes on the entire surface of leaves E (V1) and F (V2) Fig. 1(e-f). Whereas MA2VU4 has vein banding on G leaves (V1) Fig. 1(g) and vein clearing on H leaves (V2) Fig. 1(h). Various disease symptoms of P. amabilis and D. discolor induced mycorrhiza and inoculation of ORSV are shown in Fig. 1.

Disease progression: The results of this study are based on an analysis of disease incidence and disease intensity. The results of calculating the incidence of disease in P. amabilis revealed that the mycorrhizal induced and virus inoculated (MVA1) had a greater disease incidence than the virus inoculated only (VA1). In VA1 it has an average percentage of disease incidence of 54%, whereas in MVA1 it has an average percentage of disease incidence of 55.5%. Whereas in D. discolor, the treatment of virus inoculation (VA2) and the treatment of mycorrhizal induction and virus inoculation (MVA2) had the same average percentage of disease incidence, namely 58%. Calculation of the disease incidence on P. amabilis and D. discolor induced mycorrhiza and inoculation of ORSV was shown in Table 4.

The results of the analysis of disease intensity in P. amabilis showed that the virus inoculation treatment (VA1) was 26.75%, while the mycorrhizal induction and virus inoculation treatment was 32.5%. Whereas in D. discolor, the disease intensity in the virus inoculation treatment (VA2) was higher, namely 33.32%, while the mycorrhizal induction and virus inoculation treatments were smaller, namely 24.57%.

Resistance level of plants: The results of the resistance level analysis of plants on P. amabilis showed that the virus inoculation treatment (A1V) in 3 replicates was semi moderate, susceptible, very susceptible and tolerant. Meanwhile, in the mycorrhizal induction and virus inoculation (MVA1) treatment, the resistance level was very susceptible and tolerant. Overall, the results of the study showed that there were 11 very susceptible leaves, 1 susceptible leaf, 3 tolerant leaves, 1 semi moderate leaf.

Fig. 1(a-h):

Symptoms of P. amabilis and D. discolor induced of mycorrhiza and inoculation of ORSV: (a) Mild mosaic, (b) Symptomless (c) Severe mosaic and necrotic, (d) Severe mosaic and necrotic, (e) Severe mosaic, (f) Severe mosaic, (g) Mild mosaic and (h) Severe mosaic Bar 1 cm

The resistance level of plants on P. amabilis induced by mycorrhiza and inoculation of ORSV showed in Table 5.

In contrast, the results of the resistance level analysis of plants on D. discolor showed that the treatment of virus inoculation (A2V) in 3 replicates was very moderate, very susceptible and tolerant. Meanwhile, in the mycorrhizal induction and virus inoculation (MVA2) treatment, the resistance level was semi moderate, very moderate, susceptible, very susceptible and tolerant. Overall, the results showed that 3 leaves were very susceptible, 1 susceptible leaf, 7 tolerant leaves, 2 semi-moderate leaves and 3 very moderate leaves. The resistance level of plants on D. discolor induced by mycorrhiza and inoculation of ORSV showed in Table 6.

Chlorophyll (a, b and total): The results of the tukey test for chlorophyll content. In combination with P. amabilis (2.27 b) and D. discolor (1.87 b) treatments with virus induction (V) produced the same chlorophyll a (not significantly different), but the results were lower and significantly different when compared to the combination treatment between Phalaenopsis amabilis which was induced by mycorrhiza (M) 2.60 a and mycorrhizal virus (MV) 2.65 a similarly when compared with the combination treatment between Dendrobium discolor which was induced by mycorrhiza (M) 2.70 a and mycorrhizal virus (MV) 2.48 a. The results of the Tukey’s Test for chlorophyll (b, total) content in P. amabilis and D. discolor with mycorrhizal (M), virus (V) and mycorrhizal virus (MV) treatments, it was shown that Phalaenopsis amabilis and Dendrobium discolor had the same effect. Comparison results of chlorophyll (a, b, total) content in P. amabilis and D. discolor induced by mycorrhiza (M), inoculation by virus (V) and mycorrhizal virus (MV) with chlorophyll b content in control are shown in Table 7.

DISCUSSION

The OMF induction in controlling viral infection in orchids in decreasing infection symptoms and disease intensity in leaves caused by ORSV^8,13. Observation of these two orchid showed OMF Trichoderma has not been proven to be able to reduce the symptoms of infection and disease progression. Observations of symptoms of ORSV infection showed differences in symptoms of infection and incubation period in Phalaenopsis amabilis and Dendrobium discolor. Phalaenopsis amabilis showed a variety of ORSV symptoms in the form of mosaics and necrosis on days 13 and 18 after inoculation. This was in accordance with what was reported by Mahfut et al.¹ that the most common ORSV symptoms in Phalaenopsis were mosaic, necrotic, mixed necrotic and chlorotic, leaf curl and wilted leaves. In previous studies, Phalaenopsis also showed necrotic in the form of small blackish-brown circles that were concave and over time caused yellowing of the leaves^2,3,14.

In contrast to Dendrobium discolor, the average infection symptoms appear on the 18th day. Minarni et al.¹³ reported that ORSV symptoms appeared on the 11th day after inoculation in the form of leaf discoloration. Symptoms that often appear are mosaics. Several research reports on ORSV symptoms in Dendrobium, namely D. discolor showing mosaic symptoms, D. lasiantera, D. nindii, D. burana Jade×D. nindii and D. burana Mainil Wrap×D. stip in the form of a mosaic with mixed bright green and yellow spots, D. woxin and D. stratioties in the form of ring-shaped chlorotic spots and D. schulleri in the form of concentric spots. In this study, all treatments showed 3 leaves of D. discolor that did not show ORSV symptoms. Research also reported that there were several orchids that show symptomless until the end of the observation, including D. stratiotes^8,13. The difference in the symptoms of infection in these two orchids is influenced by the level of plant response to viral infection^1,3,5,8,12.

Disease development was obtained by calculating the incidence of each plant and the disease intensity of each plant. Based on the results showed variations in the incidence of the disease in each different replication. The average incidence of disease in P. amabilis is 54%, while in D. discolor, the average is 58%. Phalaenopsis amabilis has an average of 4 total leaves and 2 infected leaves, a disease incidence of 54% is obtained, while D. discolor has an average of 3 and 4 total leaves and 2 infected leaves so an average of 58 %. This means that the development of ORSV in D. discolor infects more than in P. amabilis, this is because the total number of leaves analyzed in P. amabilis is more, namely 4 leaves compared to 2 leaves in D. discolor. In this study it was shown that the incidence of disease in D. discolor was greater than in P. amabilis. The presence of mycorrhiza does not provide an effective role in reducing disease infection. In a previous study, Minarni et al.¹² reported that there was no interaction between the application of different types of planting media and doses of mycorrhizal biofertilizers on the growth and flowering of Dendrobium, this was made possible because Dendrobium sp., unable to respond well to both treatments, so that each treatment has a separate effect.

The thing that affects the calculation of disease incidence is the large number of plants infected with the virus so that the ratio of infected leaves to the total number of leaves is obtained. The genetic composition of host plants and viruses, plant age and environmental conditions before and after infection are factors that can influence the development and severity of disease symptoms. The intensity of the disease obtained shows that there are variations in the intensity of the disease. The results of this study showed that the average intensity of the disease in P. amabilis was more than 40%, i.e., there were 10 leaves showing necrotic symptoms, the rest were mild mosaic and severe mosaic. Meanwhile, the average disease intensity on D. discolor was 30%, i.e. there were 11 leaves showing mosaic symptoms. This indicated that the disease of P. amabilis was more severe than D. discolor, so the severity of each leaf increased. In a previous study, Mahfut et al.^4,5 found black-brown necrotic symptoms on the leaves of Cattleya with disease intensity between 20-80%.

The level of resistance in this study was obtained at different plant levels, namely very resistant, resistant, tolerant, susceptible and very susceptible. On average, D. discolor shows a tolerant response. In D. discolor there are also 3 leaves showing a very resistant response. Previous research by Mahfut et al.^4,5 reported that D. stratiotes and P. violacea orchids were tolerant to ORSV infection. In P. amabilis, the average resistance level is very susceptible. Several types of orchids are known to have different levels of resistance to ORSV infection. D. burana mainil wrap×D. strip, D. burana jade×D. nindii, D. woxin, O. golden shower and V. violacea showed a resistant response, while D. nindii, D. schulerii, D. kyosimori, D. liniae, P. amabilis, P. tiny white red lip×white red lip, G. scriptum and C. black lucky man×C. black lijinan pearl showed a susceptible response. Dendrobium nindii, D. discolor, D. lasiantera, D. kyosimori, D. schulerii, P. amabilis, O. golden shower and C. pandurata showed a tolerant response (P. violacea and D. stratiotes) and showed a mild response resistant (D. burana mainil wrap×D. strip, D. burana jade×D. nindii, D. woxin and G. scriptum) showed a susceptible response.

This study showed that the resistance level of D. discolor is more resistant than P. amabilis. In the D. discolor treatment, the virus inoculated (A1V) was more resistant than those inoculated with mycorrhizae and viruses. A comparison was also obtained of the level of resistance in the treatment (A1V), which was very susceptible (5): Susceptible (1): Tolerant (1): Moderately resistant (1) whereas, in the treatment inoculated with mycorrhiza and virus (MA1V) very susceptible (6): Tolerant (2). The treatment (A2V) had a comparison of plant resistance levels, namely very susceptible (2): Tolerant (4): Very resistant (2). In the treatment (MA2V2) obtained a comparison of the level of resistance very susceptible (1): Susceptible (1): Tolerant (3): Somewhat resistant (1): Very resistant (1). From the results obtained from the two treatments on both orchids it can be concluded that the presence of mycorrhizae does not play an effective role in plant resistance, because pathogenic viruses can also increase peroxidase activity as shown by Mahfut et al.^8,13 which stated that pathogen infection will increase peroxidase activity. The higher the peroxidase activity, the higher the level of plant resistance against pathogenic infections. Judging from the results obtained, more orchids were inoculated with viral mycorrhizae which showed a very susceptible response.

The results of the analysis of the content of chlorophyll a in P. amabilis and D. discolor which were inoculated with virus (V) showed a low amount. This is thought to be due to the absence of mycorrhizal roles in the formation of chlorophyll where the formation of chlorophyll requires nutrients in the form of carbon, phosphorus, nitrogen, magnesium and water which can be used in the process of forming chlorophyll a. However, although there is no Ceratorhiza, the presence of ORSV in Phalaenopsis amabilis and Dendrobium discolor, in addition to being pathogenic, is thought to induce the formation of phenols which can act as an anti-virus so that the presence of ORSV in P. amabilis and D. discolor are suspected not to damage the leaf mesophyll tissue. So it doesn't damage chlorophyll a. This conjecture was consistent with the theory put forward by Mahfut et al.^8,13 declare that ORSV enters cells and replicates so that orchid plants will activate a resistance response by forming secondary metabolites in the form of phenols. Mahfut et al.^3,11 stated that the formation of phenol can function as an antiviral. The conjectures and theories mentioned above caused the chlorophyll content in the combined treatment of both orchids with mycorrhiza (M), inoculation of viruses (V) and mycorrhizal viruses (MV) induced to be the same (not significantly different). However, when compared to the controls found in P. amabilis and D. discolor chlorophyll content was higher on both orchids induced by mycorrhiza (M), inoculation of viruses (V) and mycorrhiza viruses (MV). So it can be concluded that there is a role for Ceratorhiza induction and ORSV inoculation in P. amabilis and D. discolor. Although it does not produce resistance that is different from the chlorophyll content produced. Based on the results of the Tukey’s Test for chlorophyll b content on both orchids with mycorrhizal (M), virus (V) and mycorrhizal virus (MV) treatments was shown that both orchids had the same effect. The similarity of chlorophyll b content which has been induced by mycorrhizae (M), viruses (V) and mycorrhizal viruses (MV) is thought to be due to the formation of phenols on both orchids.

In the inoculation of virus (V), phenol functions as an anti-virus. Viruses that enter orchid leaves trigger phenol to terminate virus synthesis so that the virus cannot damage the mesophyll tissue of the leaves but the virus is still able to damage the surface of the epidermis, then shows symptoms of infection in the form of chlorotic, mosaic and necrotic. Prevention of virus entry by phenol in the leaf mesophyll keeps the leaf chloroplasts from being damaged and disturbed so that chlorophyll can be formed. This was in accordance with the theory of Mahfut et al.^8,13 which states that plants that experience pathogen infection will develop plant resistance. Pathogens can cause activation of plant defense responses in the form of hypersensitive responses. This response will induce PR-Protein so that it can stimulate lignin formation and slow down the spread of pathogens.

In the induction of mycorrhiza (M), P. amabilis and D. discolor form induced systemic resistance (ISR). The ISR itself is an increase in plant resistance that is formed due to the presence of non-pathogenic induced agents. Factors causing the formation of ISR as a result of changes in plant physiology that stimulate plant defense against pathogens through the formation of chemical compounds. This was in accordance with Mahfut et al.^8,13, who explained that the physiological changes caused by ISR can be in the form of structural modifications to the cell wall or changes in biochemical reactions in plants. Some of the changes that occur are the strengthening of the epidermis and the cortex of the cell walls as well as the formation of a barrier around the site of infection in the form of phenolic compounds to prevent the entry of viruses and prevent damage to chloroplast tissue due to viral infections.

In the induction of mycorrhizal virus (MV), P. amabilis and D. discolor formed induced systemic resistance (ISR) in the administration of mycorrhiza and formed system acquired resistance (SAR) in the administration of viruses. In this treatment, it was found that chlorophyll b did not decrease. This is because in plants that were previously infected by mycorrhiza (M) and induced resistance when orchids are induced by virus (V) the plants will be able to defend themselves by producing a hypersensitive reaction (HR). HR is a reaction that increases respiration, accumulation and phenol so that there will be anti-pathogenic and non-pathogenic which results in increased plant resistance so that plants will still be able to produce chlorophyll properly due to preventing damage to the chloroplast tissue.

The conjectures and theories mentioned above caused the chlorophyll b content in the combined treatment on both orchids with induced mycorrhiza (M), inoculation of virus (V) and mycorrhizal virus (MV) to be the same (not significantly different). However, when compared to the controls found on both orchids the chlorophyll b content was higher on both orchids induced by mycorrhiza (M), inoculation of viruses (V) and mycorrhiza viruses (MV). that’s it can be concluded that there is a role for Trichoderma induction and ORSV inoculation in both orchids. Although it does not produce resistance that is different from the chlorophyll content produced.

The results of the analysis of total chlorophyll content on both orchids induced by OMF on virus infection showed the same effect. Similar results obtained in mycorrhizal (M) inoculation, virus induction (V) and mycorrhizal virus (MV) are produced from the presence of phenol which causes viruses

that enter orchid leaves to trigger phenol to terminate viral synthesis so that viruses cannot damage tissues. leaf mesophyll but the virus can still damage the surface of the leaf epidermis and show symptoms in the form of mosaic, chlorotic or necrotic. Prevention of virus entry by phenol in the leaf mesophyll keeps the leaf chloroplasts from being damaged and disturbed so that chlorophyll can be formed. At the time of mycorrhiza (M) induction, the formation of induced resistance occurs which results in several changes occurring. These changes take the form of strengthening the epidermis and cortex of the cell walls as well as the formation of a barrier around the site of infection in the form of callus, lignin and phenolic compounds so as to prevent the entry of the virus and prevent damage to the chloroplast tissue due to viral infection. Therefore, plants will still be able to produce chlorophyll properly due to preventing damage to the chloroplast tissue^{1,5,8-10,13,14}.

The conjectures and theories mentioned above caused the total chlorophyll content in the combined treatment on both orchids with mycorrhiza (M) induced, virus (V) inoculation and mycorrhizal virus (MV) were the same (not significantly different). However, when compared to the controls found on both orchids the chlorophyll b content was higher on both orchids induced by mycorrhiza (M), inoculation of viruses (V) and mycorrhiza viruses (MV). So that it can be concluded that there is a role for Ceratorhiza induction and ORSV inoculation on both orchids Although it does not produce resistance that is different from the chlorophyll content produced^1,8,13.

According to research, there is no evidence that the induction of OMF Trichoderma increases orchid resistance to viral infections or slows the spread of disease. However, the findings of this research may provide recommendations for the kinds of orchids to be planted in the endemic regions of ORSV. This study's drawbacks include the fact that it was conducted under in vivo circumstances under various environmental variables, which prevented it from producing the intended results. In vitro research should be done more in the future to ensure that the findings support the theory. To determine the secondary metabolites produced by OMF Trichoderma in the indication of plant disease resistance, additional research was also carried out.

CONCLUSION

The OMF Trichoderma has not been proven to be able to reduce the symptoms of infection and disease progression. Based on the results obtained in the mycorrhizal induction treatment and virus inoculation showed a more severe disease infection compared to those only inoculated with the virus. The OMF could not increase resistance in P. amabilis and D. discolor, this was due to the mycorrhizal induction treatment and virus inoculation showed an average response that was very susceptible compared to the treatment that was only inoculated with the virus. The content of chlorophyll (a, b, total) mycorrhizal induction and virus inoculation treatment on both orchids was the same. In addition, there was also no difference in the level of resistance of both orchids to induction of mycorrhizal (M), inoculation of virus (V) and mycorrhizal virus (MV). This research can be used as a recommendation to determine the types of orchids cultivated in ORSV endemic areas based on the best resistance response.

SIGNIFICANCE STATEMENT

This research aims to determine the response of plant resistance through symptoms of infection, intensity of disease infection and level of plant resistance, as well as knowing the physiological response of plants through analysis of chlorophyll and carbohydrate content. The result showed OMF Trichoderma is proven to be able to prevent the appearance of symptoms of infection and disease development. The results of mycorrhizal and virus inoculations on orchids cause more severe symptoms of infection than those inoculated with viruses only. The OMF Trichoderma also has not been able to increase plant resistance in both orchids on resistance and physiological response.

ACKNOWLEDGMENTS

We thank LPPM, University of Khairun through Program Penelitian Kompetitif Unggulan Perguruan Tinggi Tingkat FKIP number 3187/PEN-PKUPT/PT/PG.12/2023 for financial support. The authors also thank University of Lampung and University of Khairun for collaboration the research.