 Oh, good morning everyone. Can you hear me well? Hello? Yes, yes, I can hear you. Okay, good. Okay, so as Luisa introduced, so today I'm going to share you guys about my experience in diagnosis of the African suture screening disease. So let's look back some background detail, some detail about some basic detail for suture screening. Suture screening is one of the most destructive diseases for the suture industry around the world. This disease is limited to sutures and a few other generals of the Drotaceous and there are currently three forms of suture screening disease. The first one is Asian form or well known as the Huolongbin HMV. This form is associated with the heat-tolerant bacteria disease, candidatus, livery bacteria, aceticus. The short name is LAS. The second form is American form. This is associated with the Americanus sebaceous short name SLAM which is less heat-tolerant than LAS and the third one is African suture screening form is associated with the heat-sensitive candidatus, livery bacteria, Africanus. The short name is LAS and all the three forms of suture screening are transmitted by the celiacs, diophorinacetrae, Gelsa aritrae, and cacopsilla cg sugar. So when infecting the plant, the suture screening can cause a number of symptoms and the most typical symptom is causing the leaf urine with blotchy motor. It also makes the leaf vein urine and the twig die back. It's called the plant growth barley with the shoot grow upright. Also make the fruit smaller, lopsided, and showing ripening color inversions where the fruit gets started in yellowing on the end near the stem instead of on the other end like in case of for the hefty fruit. At the moment all the three form of suture screening are exported to Australia, however because they are spread in so they actually potentially post the bicycle spread to Australia and we have the diagnostic assay for all of them, however only those for Asian form has been validated and used for diagnostics but not for the other two, lamb and lamb, due to we don't have the positive material for them. So for that reason in June 2019, I traveled to Nell spread in South Australia to undertake the diagnostic training for African screening at the Citrus Research International and the other purpose of my trip was also to sort the lab infected plant material to prepare the positive DNA control for variation of the visa and my trip was funded by Plant Health Australia under the diagnostic residential program. So at the first days of my training, I collected five samples to prepare the positive control. The first sample is the healthy rock lemon which is used for the negative control in my PCR. The second one is the lab infected madame vinaeus orange used at the positive control. The other three is lab infected rock lemon, lab infected carotid and the mandarin fruit, so in color in person. And for the for the adjuction for the lip I use mist ribs and for the fruit I use the pit which is the white part inside the skin and this selection was based on the report by Lee and her co-author in 2019 in phytopathology where they reported the the mist ribs and the pit have the highest concentration of the bacteria as compared to the other parts of the leaf and the fruit. Following the DNA adjustment I use the conventional PCR to detect the laugh and I expected all the four samples I collected were positive but only the sample two and the sample five were positive. The sample three and the sample four was not so the lesson from this resource is the leaf collected from the infected plant can also be negative for laugh because it sometimes it takes some time for the lab to spread to the other parts of the plant or the lab can distribute this unevenly in the plant. So to have more positive control and to bring back to a trailer so the next day I collected six more samples and this time I got on six samples positive with laugh and the most unnoticed one is the sample 11 which is I take the asymptomatic leaf from the infected plant and it's also positive with laugh so the lesson from here is the asymptomatic leaf can be also positive for laugh because when infected it takes some time for the plant to show the symptom so by the time that when we see the symptom it's too late. In my training I also have a chance to visit two orchards growing valensa oranges where they have quite a few plants have the citrus green in this disease as you can see from the picture the plants show in year in with blotchy motto and also the fruit showing the ribony color in inversion. From the field I took five four samples for testing and the samples one the sample two they are symptomatic but they test negative and the sample three and four was positive so how can we distinguish the the symptom between the negative sample and the positive sample you can see both samples so symptom yellow and symptom but for the negative sample the symptom is more symmetric which the in contrast with the positive sample the symptom more asymmetric with like yellow and blotchy like green and yellow in addition to conventional visa I also we also use the cubistar to detect the disease the cubistar is more recommended to detect for surveillance for screening and also to detect the asymmetric sample because its file is more sensitive than the conventional PCR. The first primer in the column is the universal forward primer can detect on three forms Asian American and African the second primer is the formal primer for Asian form the third primer is the formal primer for African form the next one is the forward primer for American form and only four primer were sharing the same reverse primer and the prof and if we detect this if we use the plan you can use the cost for interlocking zone and if you are screening the disease from silica you can use the windows chin and recently they proposed to replay the forward primer for Asian form by another primer it's called last 4g primer because they were it's it was found that the HIV ASF primer let missing one neurotherapy in the primer which is reduced the sensitivity of the assay so for the citrus silica this is the factor for transmitting the African disease even they look very tiny and they look very beautiful under microscopic it look the it have the wind look like the fairy tale wind and I also have a chance to see them even I'm not an entomologist so um uh to uh when when they colonize the leaf they they leave the they cost the whole on the pump on the leaf which is also the size of the present so when you see something like that you may feel that this is the child's are made present there to identify the child's are average age we can uh based on the morphology where we looking at where the is have the first branching of the four-winged vans uh jifocating uh as in the picture 1a and there's no extra cross veil uh uh as in the one we will see for the the other one is dial foreigner cg and we also looking at the head to identify the child's are where the head showing the visible uh chinocons in the picture 2b and there's no meta but this uh by c touch source first uh in the leg and also to identify the child's are you can use the dna barcoding to sequence in the coi chin and the specimen i i bring it in and i do the coi sequence and this fall in is uh through that this is a child's are or or which age uh so the outcomes from my trip um for the diagnostic skill and experiences uh the most difficult symptom for African tissue screenings disease is the leaf uh showing urine with the blotchy motor and isometric and for the fruit is showing color inversion and because it takes time for the last president to the other parts of the plant so sometimes you can get the leaf from the plant and it's that is negative which is normal and in this case you should sample the other part which is the older leaf is referred for sampling and testing uh it's also take time for the infected plant showing the symptoms so that's uh so in this case you can get the asymptomatic leaf can be that is positive so the lesson from here don't wait for the symptom don't wait to see the symptom because by the time you wait for the symptom it's too late so uh it's recommended is doing regular surveillance and testing the seamless by the time we detected them rather than testing for the plant and in this case the q pcr is before it's got it's more sensitive than the conventional pcr from the trip i also generated a number of lab positive dna control which i only bring back to australia and we have been due for validation of the pizza at pizza at size and one where one is has been that has been validated which can be for diagnosis and this can help to fill the australian diagnostic gap for the disease and also facilitating the early detection of the disease in the in the event of the disease spreading to to our country and i also bring back uh uh many specimens of the child's uh a retrace which you can use at the diagnostic rare diagnostics reference is now deposited in the herparium in our lab uh in addition to professional experience i also got some wonderful experience where i have a chance to visit the cruel national park where i actually spot many wild animals where the first time in my life so um where i can see uh to drop in power i can see uh hyena and i also can see the high the zebra walking the zyno crossing the road uh the lion walking in front of me and uh hips of elephants so uh very wonderful and okay to finish my experience i would like to thank you so much to dr queenis who is we see the person who trained me for the cc queenis and uh her staff member in ci and also thank you the plan help with trailer for my training and thank you for your listening