Report of diplodia stem rot disease in SoE-Mollo-Citrus in Timor Tengah Selatan Regency, East Nusa Tenggara

SoE-Mollo-Citrus is prominent in East Nusa Tenggara. However, a consortium of pathogens infects citrus plants, causing symptoms of broken stems with peeled bark associated with or without gum production. These symptoms could be caused by Lasiodiplodia sp. or Phytophthora sp. Considering the importance of SoE-Mollo-Citrus for society and the Government, actual and accurate information is profound regarding the presence of primary diseases that threaten it. This research aimed to determine disease intensity in central SoE-Mollo-Citrus production villages and to identify pathogens isolated from stem rot symptoms. Stratified purposive sampling method was applied to select seven Districts and ten Villages having large SoE-Mollo-Citrus populations. In each village, one orchard was randomly chosen for observation. SoE-Mollo-Citrus showing symptom of wet and dry diplodia were present in every observation village. The incidence and severity of the disease ranged from 30% in Mnelalete village to 75% in Oelbubuk village and between 21% in Tubuhue village to 51% in Oelbubuk village, respectively. Seventy percent of the collected isolates were pathogenic. Morphological and molecular identification using Primers ITS2 and ITS5 confirmed that the stem rot pathogen was Lasiodiplodia theobromae Pat. Syn. Botryodiplodia theobromae. Good agricultural practices are mandatory so this disease does not harm citrus production.


Introduction
Citrus of SoE is a favorite fruit for East Nusa Tenggara, which became known around the 1980s when SoE apple, previously a superior fruit, became extinct due to diseases.The SoE Citrus variety was released as a national superior variety named SoE-Mollo-Citrus by the Minister of Agriculture of the Republic of Indonesia in 1998.The SoE-Mollo-Citrus has been registered with the Director General of Intellectual Property of the Ministry of Law and Human Rights of the Republic of Indonesia since 2016, with ID number G 000000050.SoE-Mollo-Citrus (Citrus reticulata) has a unique combination of sweet and sour taste, orange in color with a slight green tinge, measuring 6-9 cm with a fresh fruit weight of around five to eight per kilogram.The SoE-Mollo-Citrus once won the National Horticulture Festival in Mataram, West Nusa Tenggara 2015.
Environmental conditions suitable for the growth and production of SoE-Mollo-Citrus are at an altitude of 850 -1,200 meters above sea level, which is found on the slopes of Mount Mutis, which includes four Districts, namely North Mollo, Central Mollo, Tobu, and Fatumnasi Districts in Timor Tengah Selatan Regency, East Nusa Tenggara Province.SoE-Mollo-Citrus plants require cool temperatures and tend to be dry, namely a long dry season with temperatures ranging from 28 -31 O C for growth and development.However, since 2009-2010, there has been an increase in the frequency of

Survey and sampling
The survey was conducted using the stratified purposive sampling method.Several districts were selected within the South-Central Timor Regency, and several villages were chosen within each district.The selection was based on the abundance of the soe-mollo-citrus population.In each village, one orchard belonging to the community was randomly chosen.Observations in selected orchards were carried out regarding the symptoms of diplodia stem rot disease as well as the incidence and severity of the disease.The percentage of disease severity (%) was calculated using the following formula [7], Note: I = Disease severity (%) n = Number of trees affected in a particular category v = Specific attack category score (   The survey was conducted in five Districts and ten villages as shown in Figure 1.

Isolation and identification of pathogen isolates morphologically and molecularly
The pathogens were isolated from diseased plant tissues aseptically using standard laboratory methods.The isolates were cultured in Potato Dextrose Agar media and incubated for seven days at room temperature.Identification of isolates was carried out macroscopically, namely by observing the length of time it grows to fill the Petri dish, the color of the colony, and the type of mycelium growth.Microscopic observations include the shape and color of conidia.Identification was carried out using the Illustrated Genera of Imperfect Fungi identification key [15].Molecular identification applied Conventional PCR methods that were using universal primers ITS2 (GCTGCGTTCTTCATCGATGC) and ITS5 (GGAAGTAAAAGTCGTAACAAGG) [16,17,18].Sequencing of the PCR product was done by General sequencing method using Applied Biosystem 3500 Genetic Analyzer 2500 [19].DNA sequence chromatograms were analyzed using the BioEdit program and searched for homologous sequences in the GenBank -National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST) program.

Pathogenicity test of pathogen isolates
The test was conducted on healthy and labeled SoE-Mollo-Citrus plant saplings from local seed breeders.The inoculant was a piece of pure culture of a seven-day-old pathogen isolate.The inoculum was attached to the part of the stem that has been cleaned through surface sterilization and has been injured by inserting a needle 5 times [8].The inoculum was covered with moist, sterile cotton.Observations were made regarding the time the symptoms appeared and a lesio description.

Results and discussion
The ten villages where the research's observation sites as shown in Figure 1 were SoE-Mollo-Citrus production hub.Fatumnasi District is located at about 1480 meters above sea level.It was located at the highest altitude among the seven surveyed Districts, followed by North Mollo District (approximately 1,007 meters above sea level), Central Mollo District (944 meters above sea level), Tobu District (at 867 meters above sea level), South Amanatun District (at 856 meters above sea level), West Amanuban District (at 814 meters above sea level), and Kie District (at 776 meters above sea level) [20].

Symptoms of diplodia stem rot
The results of observations showed that diplodia stem rot disease was found in all villages at the research locations.Symptoms of dry diplodia stem rot disease were found in all villages.In contrast, symptoms of wet diplodia were found in seven of the ten research location villages, namely the villages of Mnelalete (West Amanuban District), Oelbubuk (Central Mollo District), Netpala and Ajaobaki (North Mollo District), Tune (Tobu District), Tesi Ayofanu (Kie District) and Kokoi village (South Amanuban District).Symptoms of diplodia stem rot appear on the main stem, primary branches, and secondary branches.The stems cracked, and the bark peeled off.Some of these symptoms produce gum or symptoms of "wet diplodia," and some did not have gum or symptoms of "dry diplodia" (Figure 2).

Disease incidence and severity
The incidence of diplodia disease includes the number of plants with symptoms of wet diplodia and dry diplodia counted in one orchard or surveyed area, ranging from 30% in Mnelalete village (West Amanuban District), to 75% in Oelbubuk village (Mollo Tengah District).The severity of diplodia disease ranged from 21% in Tubuhue village (West Amanuban District) to 51% in Oelbubuk village (Central Mollo District) (Figure 3).Damage symptoms were found on the main stem, primary and secondary branches.These data indicate that stem rot disease can destroy SoE-Mollo-Citrus commodities if prevention and effective disease control are not taken seriously.Apart from diplodia disease, citrus also suffer from huanglongbing disease caused by bacteria.Zhao et al [21] and Zhen et al. [22] showed that the interaction between Lasiodiplodia sp. and huanglongbing disease may exacerbate preharvest fruit drop, thereby worsening the quantity and quality of the citrus harvest and even increasing post-harvest damage.

Isolation and identification of pathogenic isolates
Two isolates were isolated from each village, therefore 20 isolates were obtained.The colony morphology of all isolates showed similarities in terms of colony growth speed and colony color.The growth of the isolate was relatively fast.The isolate grew to fill a Petri dish with a diameter of 9 mm on the fifth day.Initially, the mycelium was white.On the fifth day, it became dark greenish black.On the seventh day, it became black (Figure 4).This description is in line with what was reported by Henuk [8] that L. theobromae was a fast grower, having oblong or oval conidia.Immature conidia were 1-celled and hyaline, while mature conidia were 2-celled with transverse septa, dark or black.Conidia were observed from isolates that had been incubated for 10 -14 days.Conidia tend to form on old isolates.Conditions of limited nutrition trigger fungi to produce reproductive organs to survive.In addition, the type of growing media also affects the quantity of reproductive organs like pycnidia in the M. phaseolina fungus [23,24].The conidia were light to blackish brown, oval with blunt ends at both ends, and some had horizontal septa in the middle of the conidia, but some were not partitioned.Old conidia were generally darker in color and have partitions or septa [25].This study observed two conidia groups: conidia formed asexually in the anamorph phase and conidia formed in the sexual or teleomorph phase.Conidia in the teleomorph phase appear to form in groups of eight conidia in one sac called the ascus (Figure 5a).The asci were formed in the pycnidia fruit body (Figure 5b).
The isolate in the teleomorph phase was Botryosphaeria rhodina (Berk.& M.A. Curtis) Arx, while the isolate in the anamorph phase was Botryodiplodia theobromae Pat.[8].However, along with developments of fungal taxonomy, based on THE AMSTERDAM DECLARATION ON FUNGAL NOMENCLATURE, it was agreed to name one fungus with one name known as "One Fungus = One Name" [26].The accepted name for Botryodiplodia theobromae and Botryosphaeria rhodina is Lasiodiplodia theobromae (Pat.)Griffon & Maubl.Botryodiplodia theobromae and Botryosphaeria rhodina are now recognized as synonyms of Lasiodiplodia theobromae.
Molecular identification confirmed the morphological identification result.The sequence of sampled isolates from Fatumnasi village showed a similarity of 99% to 100% to several kinship nearby fungi, as shown in Table 2. Fatumnasi isolate has the identity of 100% similarity to three accessions of Lasiodiplodia theobromae (accession number ON059601.1;MW341584.1;and MW341584.1),and 99,59% similar to one accession of Lasiodiplodia theobromae (accession number MW138048.1).

Pathogenicity test of fungal isolates
The pathogenicity test showed that out of the ten isolates from each village tested, seven isolates were pathogenic.SoE-Mollo-Citrus saplings inoculated with isolates from Tubuhue, Oelbubuk, Ajaobaki villages began to show symptoms on the 11 th day after inoculation.Isolates from Tune and Fatumnasi villages started showing symptoms on the 9 th day, and isolates from Tesi Ayofanu and Kokoi villages started showing symptoms on the 12 th day after inoculation.Initial symptoms appear as yellowing and slight brown spots measuring around 3-5 mm at the upper stem joints, then develop into small cracks in the bark (Figure 6a, 6b, 6c), and some produce gum (Figure 6d).
Differences in the incubation period might indicate differences in virulence and differences in strains.More virulent strains will infect more quickly and produce larger lesions.Severity of infection by L. theobromae was controlled by LtScp1 gene that prevents the fungus from degrading during infection [27], as well as by PR genes, including putative QDO, cell wall-degrading enzymes, sugar transporters, kinases such as the histidine kinases, peptidase, carboxypeptidase, and putative cytochrome p450 protein [28].This verifies that Lasiodiplodia theobromae is an important pathogen that may threaten SoE-Mollo-Citrus's population and production.

Figure 2 .
Figure 2. Symptoms on SoE-Mollo-Citrus similar to the typical symptoms of diplodia disease caused by Lasiodiplodia theobromae.(a) Habitus of diseased SoE-Mollo-Citrus citrus plant; (b) Symptom of dry diplodia, stem rot and peeling of bark without gum; (c) Symptom of wet diplodia, stem rot and peeling of bark accompanied by gum.

Figure 4 .
Figure 4. Colony of Lasiodiplodia theobromae isolated from SoE-Mollo-Citrus.(a & b) Fifth-day isolate; (c & d) seventh-day isolate pictured from the upper and bottom of Petri dish.

Figure 5 .
Figure 5. Conidia of Lasiodiplodia theobromae Pat (a) Teleomorph conidia formed in groups of eight inside the ascus.Immature conidia were hyaline while mature conidia were dark; (b) Picnidia containing asci; (c) Anamorph conidia having septa in the middle; (d) Aseptate anamorph immature conidia.Examination was done using light microscopy with 400 magnification.

Figure 6 .
Figure 6.Pathogenicity test on SoE-Mollo-Citrus plant saplings.Symptoms of yellowing and small brown spots around 3-5 mm at the top of the stem joint develop into small cracks in the bark (a-c); gum that appear in the necrotic area (d).

Table 2 .
Sequences similarity of Fatumnasi isolate from SoE-Mollo-Citrus and kinship nearby fungi