Develop an ecient inoculation technique for Fusarium solani pathogeny assessment in Phalaenopsis orchids

Background: Phalaenopsis is one of the important ornamental plants worldwide. It plays the most signicant role in ower exportation in Taiwan. However, the yellow leaf disease caused by Fusarium spp. has reduced the orchid ower yield 10-50% yearly. Varieties resistant to yellow leaf disease associated with Fusarium is urgently needed for orchid growers and breeders, and is the ultimate solution for the long-term goal. To achieve this, phenotyping is the rst step and the most necessary information for further studies, such as resistance gene identication, quantitative trait loci identication, and genome-wide association study. Results: The inoculation of Fusarium was performed in either abbreviated stem or detached leaf, and the pros and cons were compared. The former is the general method of phenotyping for estimating the tolerance to yellow leaf disease of Phalaenopsis, but it is time-consuming and spacy, and thus not suitable for the assessment of large numbers of samples. In contrast, the latter not only showed a similar trend of disease severity with time reduced to only one fourth of the former one but also less space needed. Conclusions: This solution allows a better phenotyping approach for the fast detection of yellow leaf disease associated with Fusarium in a large number of Phalaenopsis samples.


Background
Orchids are important owers for horticultural production and exportation worldwide. Among them, Phalaenopsis app. are the most popular and signi cant orchids. In Taiwan, the export value of Phalaenopsis in 2019 was about 140 million US dollars which covered 75% of the total value of orchid exportation (https://www.coa.gov.tw/). Fusarium spp., pathogens that damage crop widely and cause up to 50% yield loss in soybean, banana, tomato, and wheat (Perincherry et al., 2019). For orchids, symptoms developed after infection by F. solani include leaf spots, leaf blight, sheath rot, and root rot. The infection spread worldwide because of international trade and monoculture (Swett and Uchida, 2015).
Fusarium solani, with host speci city of Phalaenopsis has been found in Taiwan (Chung et al., 2011), Korea (Kim et al., 2002), Hawaii (Swett and Uchida, 2015), and Australia (Laurence et al., 2016). Infection by F. solani causes a reduction of 20-30% yield every year in Taiwan (Su et al., 2010). To suppress the activity of Fusarium, 16 fungicides have been tested for growth inhibition of F. solani. However, none of them show signi cant effects on the reduction of the damage caused by F. solani in the greenhouse environment (Su, 2014), or after a long distance of shipment from country to country for several months (Liao, 2012), although they showed great inhibition rate on conidiospore germinations with different diluted concentration (Su, 2014). Instead of chemical treatment to reduce the infection rate of Fusarium on Phalaenopsis, development of resistant varieties with quantitative treat loci (QTLs) resistance to F. solani via breeding program is an alternative. The resistance genes or QTLs to Fusarium spp. have been identi ed from Arabidopsis (Diener and Ausubel, 2005), wheat (Buerstmayr et al., 2002) and pea (Coyne et al., 2015). However, so far no reports of Fusarium resistance QTL have been reported in orchids. Phenotyping symptoms after pathogen inoculation is the most important step for the identi cation of resistance genes or QTLs. A long duration of 4-6 weeks for disease progression after inoculated at the abbreviated stem of live Phalaenopsis plants have hampered the assessment. Even worse is that large space is needed for examination enough plants for Phalaenopsis-Fusarium study (Su et al., 2012).
In this study, a modi ed method for assessment of the yellow leaf symptom infected by F. solani in detached leaf was established with reduced observation time of 6 days and much less space needed. In addition, the present study showed that similar trends of symptom development were observed for the infection of F. solani between the detached leaf and the abbreviated stem. Furthermore, symptom detection based on the detached leave method was stable and feasible for examining a large quantity of plants.

Sample Collection
Five Phalaenopsis cultivars including TAI_A2945, TAI_A7403, TAI_A9168, TAI_A10040, TAI_A10746 from Taida Orchid Nursery were used in this research, to t the real transportation situation, twenty mericlones cultivated were provided from orchid nursery, each individual was planted in 2.5-inch pots for 2-3 months which close to the true plant size for oversea production. Twenty individuals of each cultivar were used in each replicate and separated equally for Fusarium inoculation to detached leaf and abbreviated stem. Three replicates in total were performed with one-week interval among each replicate. Before the inoculation, the detached leaf was prepared from one mature leaf of each individual which cut around 8.5 cm from the apexes and then xed the detached leaf in a 15 cm diameter dish. We kept all plant materials in a room with the constant temperature at 27 °C for two days before the detached step to make sure all plants started with the same environment.

Fusarium inoculation
The procedures of inoculation for two organs were performed as previously described by Su et al. (2012).
Brie y, seven wounds arranged in a circle was created before inoculation. The pathogenic fungi, Fusarium solani, was provided from Taiwan Agricultural Research Institute isolated from the orchid nursery in Taiwan. The spore suspension was diluted to 10 3 pfu/ml for detached leaf inoculation and 10 5 pfu/ml for the abbreviated stem inoculation. After inoculation, detached leaves, and abbreviated stem were incubated in climate control room (HiPoint, EH-1800) set at 27 ± 1℃ with 100% humidity. The photo of each detached samples was taken from the second days after inoculation to sixth days after inoculation. Besides, images of each abbreviated stem samples were taken from the second days after inoculation until the symptom rank 9 showed in any tested cultivars.

Symptom Ranking
To distinguish the degree of pathogen resistance from all inoculated detached leaves or abbreviated stems, the disease severity level (DSL) was ranked everyday after inoculation for 6 days (detached leaf) or more than 3 weeks (abbreviated stem). Disease severity index (DSI) was then calculated with the following formula (Chiang et al., 2017).

Results
Pre-exam to distinguish the DSL ranks for both detached leaf and abbreviated stem A pre-exam was performed to distinguish the different ranks of DSL by inoculating Fusarium on the detached leaf and the abbreviated stem. Variety TAI_A10746 was selected to show the scale of DSL mainly, except the rank 10 of detached leaf (variety CH151, Fig. 1). The DSL was ranked as followed: Level 0: no symptom; level 1: number of black holes less than 3; level 2: number of black holes between 4 to 7; level 3: black holes connect together; level 4: black lesion spreads outside of the wound; level 5: less than 3 holes lled by the hypha from F. solani; level 6: 4 to 7 holes lled by hypha from F. solani; level 7: the hypha mixed together; level 8: yellow symptom emerges; level 9: yellow symptom diffuses to an entire detached leaf or abbreviated stem; level 10: the red spores show (Figs. 1 and 2). These DSL levels were applied to the photos we took from all ve varieties and three replicates for calculating the DSI of different tissues.

Similar trends of symptom development from detached leaf and abbreviated stem inoculated with F. solani
According to previous test, cultivar TAI_A7403 and TAI_A10040 have been shown to be highly tolerant and susceptible to F. solani infection. We rst checked whether the symptoms on detached leaf and abbreviated stem were similar after inoculated F. solani. First of all, the symptoms of tolerant and susceptive cultivars that developed on the detached leaf and abbreviated stem were shown. There was no symptoms in the detached leaf and abbreviated stem of variety TAI_A7403 (Fig. 3A, B, E, F), but it developed strong yellow leaf symptoms in both the detached leaf and the abbreviated stem of variety TAI_A10040 (Fig. 3C, D, G, H). Second, a similar disease severity index pattern among ve tested varieties were shown between detached leaf and abbreviated stem (Fig. 4). Highly positive correlation detected between detached leaf and abbreviated stem among replicates, as the R square was 0.96, 0.91, and 0.95 for replicate 1, 2, 3, respectively, and p-value lower than 0.05 of all replicates (Table 1). leaf has the highest correlation coe cient 0.96 between replicate 2 and 3, and the lowest correlation coe cient 0.84 between replicate 1 and 2 with the p-value 0.0081 and 0.0771 respectively. On the other hand, the correlation among replicates from the abbreviated stem was higher than 0.9 with p-value 0.0292 (replicate 1 vs replicate 2), 0.0132 (replicate 1 vs replicate 3), and 0.0299 (replicate 2 vs replicate 3) (Table 2). In addition, symptom observation time is also an essential point to be considered before screening a large number of samples or varieties. In our study, it took more than 20 days for abbreviated stem (ranged from 21 to 32 days) but it took only one week (6 days) for the detached leaf to distinguish the differences among varieties after infection with F. solani (Table 3).

Discussion
Detached leaf is a better plant material for assessment of DSI upon F. solani inoculation A good pathogeny assessment technology may consider the following characters: repeatability, sensitivity, and observation time. Different techniques of Fusarium inoculation has been tested in maize ear to nd out the best way for evaluating a large number of maize genotypes (Clements et al., 2003). In our study, high correlations detected among all replicates from both organs (Table 2) indicating the trusted repeatability. In addition, the severity pattern from two organs was highly correlated among replicates (Table 1). A similar severity pattern after Fusarium inoculation between organs is observed from ear and silk in maize as well (Reid et al., 2002). This nding indicates that the severity rank obtained from the detached leaf well represents the rank detected from the abbreviated stem. Furthermore, the detached leaf showed a wider DSI range among tested varieties than the abbreviated stem, displaying the stronger sensitivity from detached leaf than abbreviated stem. Regarding the observation time, more than 20 days were needed for symptom development after inoculation in the abbreviated stem, similar to a previous study that took 4 weeks for symptom detection in orchids (Su et al., 2012). In contrast, 6 days after inoculation with F. solani was enough to distinguish the ranges of symptoms from detached leaf, indicating one-fourth time of inoculation in the abbreviated stem. Last but not least, the space requirement is also an important issue. It was less spacy for the detached leaf as compared to that for the abbreviated stem. A dish with 15-cm diameter and 2-cm height are enough for one sample of the detached leaf, but for the abbreviated stem, a living plant in a 2.5-inch pod is requested. From all advantages mentioned above, inoculation in the detached leaf is reliable, more sensitive, shorter time consuming and less experiment space needed than the inoculation in the abbreviated stem, which could be the better solution for Fusarium assessment in Phalaenopsis orchids.

Examining the biochemical defense of Phalaenopsis-Fusarium interaction
For the defense of the invasion from pathogens, plants develop both structural and biochemical mechanisms to protect themselves. When Fusarium spp. infect plants, it stars to destroy the structural defense, and then override the plant biochemical defense by producing host-speci c mycotoxins