Identification of Pathogens Causing Bulb Rot Disease on Garlic (Allium sativum L.) in Central Java, Indonesia

Garlic bulb rot disease was found from garlics (Allium sativum L.) cultivated from 2017 to 2019 by farmers in Central Java Province, Indonesia. The initial symptoms of the disease were stunted, leaf yellowing, and necrotizing to rotten bulbs. This research was conducted to determine the major causal agent of garlic bulb rot disease in Central Java. A survey was carried out in five regencies across Central Java that were major garlicproducing areas. Nematodes were isolated using water immersion methode and pathogenic fungi were isolated on Potato Dextrose Agar (PDA). Nematode identification was carried out based on the Ditylenchus dipsaci morphological and morphometric character. Seven isolates of Fusarium species were obtained from infected garlic. Identification of four chosen isolates were performed by sequencing the TEF-1α gene. The TEF sequence of isolate TB3, KK1, and KK4 showed 99% similarity with several F. oxysporum, BT3 sequences showed 98% identity with several F. solani, and all were deposited in the NCBI GenBank. Three locations were positively infected by the interaction between D. dipsaci and Fusarium sp. Based on the results of the morphological identification, parasitic nematode was identified as D. dipsaci, while based on the morphological and molecular identification isolates Fusarium were identified as F. oxysporum and F. solani, respectively, as first report causal agents of garlic bulbs rot in Central Java.


INTRODUCTION
Garlic is an economic crop cultivated in Indonesia. Bulbs rot disease was found in garlic crop in Central Java, Indonesia (Indarti et al., 2018). The infection of bulb to rot disease in the field causes bulbs to be not sellable. Yield loss caused by bulb rots disease can reache 35-40% (L.-J. Zhang et al., 2017). The disease infect bulbs and root of garlic plants. Symptoms include leaf yellowing and premature death. The initial symptoms of bulb rot disease are shrunken bulbs that become softer, darker and eventually decay. Bulb and root rot are caused by Ditylenchus dipsaci and its occurrence have been reported in Canada (Hajihassani & Tenuta, 2016), Israel (Aftalion & Cohn, 1990), Mexico (French et al., 2017), Ohio (Testen et al., 2014), Turkey (Yavuzaslanoğlu et al., 2015), and Indonesia (Indarti et al., 2018). Garlic bulb rot can also be caused by the infection of fungal pathogen, such as from the genus Fusarium (Dugan et al., 2003;Moharam et al., 2013;L.-J. Zhang et al., 2017).
Interaction between pathogens and other organisms associated with plants can increase the severity of a disease. The association between Meloidogyne incognita with Fusarium clamydosporum can aggravate the disease (Devappa et al., 2009). Several cases of plant-parasitic nematode associations with fungi have been reported, such as the pathogenic relationship between D. dipsaci and Fusarium oxysporum f. sp. medicaginis that can break the resistance of varieties with nematode and Fusarium -resistant characteristics in alfalfa (Griffin, 1990). The synergistic interaction between D. dipsaci and Rhizoctonia solani were also reported in sugar beets (Hillnhütter et al., 2011). Other association between M. incognita and F. oxysporum in tomato plants caused complex diseases (Kassie, 2019). Therefore, investigation on the causal agents of bulbs rot disease was done and the role of both nematode and fungi were determined and the possible interaction between both of them in bulb rot disease incidences.

Collecting Samples from Infected Garlic
Samples of infected garlic were collected from garlic producing area in Central Java (Magelang, Temanggung, Karanganyar, Tegal, and Brebes) during October 2018-April 2019. Bulbs showing rot symptoms were recorded. Furthemore, samples of infected plants were taken to isolate pathogens on PDA plates. Nematode samples were analized from bulbs and infected roots.

Isolation and Identification of Pathogens
Both nematodes and fungi were isolated from infected bulb, mainly from garlic bulbs showing rotting discoloration or wilting. Nematode extraction was done using the water immersion method (S.L. Zhang et al., 2014). Morphological and morphometrical observation of each nematode specimens was done by comparing to related published data for identification to species level. Fusarium collected from rotten bulbs were sterilized in 5% NaOCl solution for 30 seconds, rinsed with sterilized water for five times. Pieces excised from lesion margins were transferred to potato dextrose agar (PDA). Cultures were incubated at 25 o C in dark conditions for seven days.
Fungal colonies were purified either by using successive transplanting of the colony edges or by using single spore techniques. The purified fungi were identified according to the fungal morphological and microscopical characteristics as described by Leslie and Summerell (2006). DNA extraction was carried out as described by Zhu et al. (2014). Genomic DNA from Fusarium isolates were used for PCR amplification of the TEF-1α gene using the primer EF-1 (forward primer; 5'-ATGGGTAAGGA(A/G) GACAAGAC-3) and EF-2 (reverse primer; 5'GGA (G/A)GTACCAGT(G/C)ATCATGTT-3') following conditions described by O'Donnell et al. (1998). The products were sent to Apical Scientific Sequencing, 1 st BASE DNA Sequencing. The sequences obtained were assembled and edited manually using BioEdit v. 7.0.9 (Hall, 1999), and then analyzed using Basic Local Alignment Search Tool (BLAST) NCBI to clarify the homology from closest species. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model (Tamura & Nei, 1993). The tree with the highest log likelihood (-1861,45) was shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 21 nucleotide sequences. There were a total of 689 positions in the final dataset. Evolutionary analyses were conducted in MEGA (Molecular Evolutionary Genetics Analysis) software version X (Kumar et al., 2018). Athelia rolfsii was used as an outgroup for the analysis. The obtained culture isolates were maintained on PDA plates and kept in refrigerator at 5°C for further study.

Pathogenicity Test
The pathogenic relationship on plant parasitic nematodes as an opening agent for fungal infection was tested (Griffin, 1990;Hillnhütter et al., 2011;Kassie, 2019). In this study F. oxysporum was chosen for the pathogenicity test because it was found in the field and have been reported to interact D. dipsaci. To study the effect of simultaneous or sequential infestation. on garlic plant was designed under the greenhouse and maintained at 23 ± 4 o C. Plants were inoculated with adding 300 D. dipsaci per pot and 5 × 10 7 microconidia of Fusarium oxysporum. Inoculation of all pathogens were applied singularly or in combination. Treatments were 1) uninoculated (control), 2) inoculation of D. dipsaci three days before inoculation of F. oxysporum, 3) inoculation of F. oxysporum three days before inoculation of D. dipsaci, 4) inoculation of D. dipsaci and F. oxysporum simultaneously, 5) inoculation of D. dipsaci alone, 6) inoculation of F. oxysporum alone. Treatments were replicated 5 times, 15 plants were used for every replication. The experiment was terminated when plants reached 8 weeks-old, plant showed mortality, and bulbs and root weights were measured. Arifin et al.: Identification of Pathogens Causing Bulb Rot Disease on Garlic (Allium sativum L.) in Central Java Severity of above-ground symptoms were periodically assessed for each plant on a 0-6 scale, according to the percentage of foliage with yellowing or necrosis: (0 = 0%, 1 = 0.1-19.9%, 2 = 20-39.9%, 3 = 40-59.9%, 4 = 60-79.9%, 5 = 80-99.9%, 6 = dead plant). Disease incidence (D.I.) was calculated according to Devappa et al. (2009) as: Where, (n) = the number of diseased transplants per category, (v) = category number, N = total number of the transplants, (V) = maximum of category. Reisolation was carried out from infected transplants showing disease symptoms. The nematode and the isolated fungi were compared with the original causal agents cultures used.

Statistical Analyses
Plant growth parameters were subjected to analysis of variance with the general linear models procedure of SAS (SAS Institute, Cary, NC). Differences between root and bulb weights of the treated plants and the untreated (control) plants were determined by using Tukey's test at P < 0.05 (SAS Institute, 1998).

Sampling and Diagnosis
Five different district location of Central Java were surveyed and plants along with the soil surrounding them were collected. In total, 94 plant samples exhibiting wilt symptoms were collected from 21 fields in five districts (Table 1). Results from three districts implied interaction between D. dipsaci and Fusarium spp. on garlic bulb showing rot disease. Garlic bulb rot in the field showed specific symptoms ( Figure 1). Symptoms could be observed on leaves, bulbs, and roots of plants since the vegetative and through the generative phases. Chlorosis were observed on leaves. Wilting from the top to the bottom of the plant were also observed. Infected plants were usually easily uprooted from the ground. Early symptoms of this disease were wilted plants and defected growth on roots and tubers of plants. As the disease progresses, the plant bulbs would turn brown and eventually rot ( Figure 1c). Yin et al. (2015) reported that the initial symptoms of bulbs rot disease were stunted plant growth, chlorosis in apical leaves, plants withering and dying. However, field symptoms caused by nematode showed  Zhang et al., 2017;Indarti et al., 2018).

Nematode Identification
Garlic samples tested came from five districts in various garlic cultivars. Detection results showed that from samples of positive garlic bulb rot disease confirmed the presence of D. dipsaci. D. dipsaci morphological characters had stylets with a length of 7.81-12.42 μm. Knob stylet were rounded and well developed. Median bulb were muscular, with thickening of lumen walls and clearly observable. Basal bulb were not overlapping with the intestine (Figure 2). Tail terminus was pointed. The morphological character of this nematode followed the criteria of D. dipsaci (Mai et al., 1968;International Plant Protection Convention [IPPC], 2015;EPPO, 2017). Baicheva and Budurova (1994) reported that the morphological character of D. dipsaci were slim bodies, elongated then tapered body shape, thin and flat nematode lip, clear cuticle annulation, short stylet, short nematode, female nematode had monodelphic vulva, male nematode had a covering that covers bursa until ¾ the tail length, and at the end of the tail was tapered. It had a median bulb shaped starting from its ovoid to fusiform, with an esophagus that does not overlap , with tail length of 4-7 times its body width (IPPC, 2015). Morphometric measurements for D. dipsaci females (n=5) were ± 0.50 (0.35 to 0.62) mm for body length, max body length to body width of ± 30.97 (25.82 to 41.60) µm, body to oesophageal length of ± 5.24 (3.88 to 9.08) µm, and body to tail length of ± 9.13 (7.66 to 12.32) µm. Referring to Indarti et al. (2018) the morphometric values complied with the criteria of D. dipsaci. Morphological characters resembled as D. dipsaci. Yavuzaslanoğlu et al. (2015) reported that this nematode infestation caused yield losses to 15% in onions and high intensity attacks could even reach 90% loss. The discovery of D. dipsaci in Central Java supports the report of Indarti et al. (2018) about the first detection on the discovery of this nematode in Temanggung.

Identification of Pathogenic Fungi
A total of 7 fungal isolates (TK3, TT2, TK4, KT2, KK1, KK4, BT3) were collected from infected garlic crops. Three isolates (KT2, KK1, KK4) were found from Magelang district, 3 isolates (TK3, TT2, TK4) from Temanggung district and 1 isolate (BT3) from Brebes district ( Table 1). The colony of all isolates had similar appearance. The colony color of each isolate on the PDA medium showed to initially have white mycelium that then remained white for TT2 and KT2 isolates, turned purple for TK3 and TK4, or pale purple for KK1 and KK4 (Figure 3). Different fungal pigmentation occurs because its growing medium. Wiemann et al. (2009) reported that some pigments have a protective role against environmental stresses such as irradiation and oxidation while others contribute to virulence. Therefore all isolates could not clearly be differentiated.
The bottoms surface of the petridish, concentrated color could be found from isolates TK4, KK1 and KK4. Ignjatov et al. (2017) suggested that the Fusarium sp. colonies are sometimes white but more often slightly purple, where as according to Summerell et al. (2003) most of the isolates of Fusarium sp. had colonies that were white or accompanied by purple or pink coloration at the center of the colony. This was consistent with findings from Kassie (2019) that reported F. oxysporum on PDA media forms a mycelium which is insulated and initially white and gradually turns to purple. The occurrence of color differences over time is more likely due to the accumulation of colors produced during the growth of F. oxysporum (Ambar et al., 2019). Based on observations made on each tested isolate, in general the isolates produced abundant air cotton-like mycelium, white in color and sometimes slightly purple.
Microconidium observed under the microscope was generally round or oval, oval and sometimes curved if it had septae, with sizes ranging from 2.23 × 2.20 µm -14.87 × 4.83 µm ( Figure 5) Microconidia were always present, especially those that do not act as a result of monophialid or conidiophores with short branches or false heads ( Figure 6). F. oxysporum had conidiophores (monofialid) with a short stem which is bound at 3-5 microconidium bound (Palmero et al., 2012). This was supported by the statements of Leslie and Summerell (2006) that stated that the length of the F. oxysporum phialid is shorter than that of F. solani.
Based on the colony's morphology and characteristics of macro and micro conidia, seven fungal isolates were identified as Fusarium. After further microscopic observations, six isolates were identified as F. oxysporum based on the macroconidia characteristics which were thin walled with 3-5 septate, while based on false head characteristic, one isolate was identified as F. solani. Molecular identification are more accurate and can be used as a comparison method between morphological observation methods.  Molecular identification was done on 4 isolates based on morphology and sampling site. The four chosen isolates (KK1, KK4, BT3, and KT2) were sequenced for the EF-1α gene. The results using EF1 and EF2 primers, which were specific primers of the Fusarium genus, showed DNA bands of 750 bp. O'Donnell et al. (1998) produced DNA bands with molecular weights of 700 bp when using this specific primer for PCR method. The TEF sequence of isolate TB3, KK1, and KK4 showed 99% similarity with several F. oxysporum sequences. Sequence of BT3 showed 98% identity with several F. solani and they were deposited in the NCBI GenBank. Phylogenic trees were arranged based on the results of DNA band sequencing. TEF sequences of Fusarium genus were aligned with the consensus region using CLUSTAL W program and 1000 bootstrap replicates were used on sequence analysis. Three isolates (KK1, KK4, and KT2) were compared to the reference F. oxysporum strain (FJ538245.1; JQ965439.1; JQ965441.1; JQ965444.1; KX6097 08.1; MK651510.1; MK266491,1; MH341212.1; MG356947.1). High degrees (94%) of phylogenetic similarity was detected (Figure 7). The results were consisted with morphological result of this study implying that Fusarium isolated from infected garlic bulb in Central Java were F. oxysporum and F. solani.

Mix Symptom of Ditylenchus dipsaci and Fusarium spp. in Garlic Crop
Three of the five districts had D. dipsaci and F. oxysporum. D. dipsaci infection showed dwarfed plants or yellow leaves with twisted leaf tips. While infection of both D. dipsaci and F. oxysporum showed specific symptoms of yellowing of leaves and necrotic to rotting tubers on bulbs. Interaction between pathogens and other organisms associated with plants can increase the severity of a plant disease (Meena et al., 2016).
Association between nematodes and pathogenic fungi may cause far greater infections than only one of the pathogens (Mudawi et al., 2018). Association between plant-parasitic nematodes and pathogenic fungi caused severe damaged on plant tissues. Plant parasitic nematodes create openings for fungal infection (Wiratno et al., 2019). This was indicated by garlic bulb showing rot and collected from Tegal which only found the presence of D. dipsaci with dwarf plant symptoms. In contrast, Magelang samples possessed D. dipsaci and Fusarium sp. and showed severely damaged plants with yellow leaves and bulb rot.
Development of disease symptoms is not only determined by the role of pathogens, but also depends on the complex interrelationships between host plants, pathogens, and prevailing environmental conditions, and for soil-borne pathogens will be largely determined by their interactions with other microorganisms that occupy the same ecological niches. Endoparasitic nematodes, such as Ditylenchus, will penetrate roots through the root tip which is still actively growing and damage caused by nematodes determines the severity of disease by soil-borne pathogens such as Fusarium (Back et al., 2002;Williamson & Gleason, 2003). According to Hillnhütter et al. (2011), D. dipsaci penetration into plant bulbs will result in injuries that occur mechanically and chemically causing easier infection of fungi.

Pathogenicity Tests
The role of D. dipsaci and F. oxysporum as a cause of bulb rot disease, was tested by inoculating D. dipsaci and F. oxysporum on garlic plants. Infected plants and disease intensity on leaves caused by D. dipsaci and Fusarium spp. are shown in Figure 8.
L.-J. Zhang et al. (2017) reported that the initial symptoms of bulb rot disease were stunted plant growth, chlorosis on apical leaves, plants withering and dying. However, field symptoms caused by nematode had different or uncertain characteristics. Symptoms could be single symptoms or a combination of several symptoms including dwarf plants or leaf chlorosis or necrosis in bulbs (Indarti et al., 2018).
Single inoculation using Fusarium significantly affected the bulb weight, but D. dipsaci inoculation alone showed no significant difference compared to the control. Single inoculation treatment of D. dipsaci showed that bulb weight was not significantly different from the single inoculation treatment of F. oxysporum. However, the bulb weight values indicated that single inoculation of F. oxysporum resulted in higher yield. This was presumably because D. dipsaci Central Java based on PCR analysis using primers EF1 and EF2 could not multiply optimally due to suboptimal temperature at the study site. This was supported by Indarti et al. (2018) who stated that the abundance of D. dipsaci populations was positively correlated with soil temperature. Higher population abundance of D. dipsaci were found in soil with lower temperature. Disease intensity of infected plants caused by D. dipsaci and Fusarium spp. are shown in Table 2. The results of this study revealed that D. dipsaci and F. oxysporum could damage singularly (inoculation D. dipsaci alone or F. oxysporum alone) or simultaneously. The presence of D. dipsaci and F. oxysporum has been reported as a cause of garlic rot disease (Testen et al., 2014;French et al., 2017;L.-J. Zhang et al., 2017;Indarti et al., 2018). The disease intensity due to combination of inoculation treatments showed significant difference compared to the control (Table 2). Disease intensity of single treatments (inoculation of D. dipsaci alone or F. oxysporum alone) were significantly different from the inoculation of D. dipsaci and F. oxysporum together. The disease intensity of D. dipsaci inoculation followed by all treatments did not show significant differences. Inoculation of D. dipsaci and F. oxysporum together resulted in the highest disease intensity compared to other treatments. This shows D. dipsaci and F. oxysporum synergistically increase the intensity of bulb rot disease. Following the report of Pujiastuti et al. (2014) who stated that the synergistic interactions of the combined pathogen Meloidogyne spp. and F. oxysporum in garlic increase the intensity of wilting symptoms of basal rot disease.

CONCLUSION
Garlic bulb rot in Central Java was caused by D. dipsaci, F. oxysporum, and F. solani complexes. The association between D. dipsaci and F. oxysporum increased the intensity of bulb rot diseases by up to 98% in garlic plants.