Pathogenic Variability of the Jackfruit-Bronzing Bacterium Pantoea stewartii Subspecies stewartii Infection to Jackfruit Varieties and Its Pivotal Plant Hosts in Malaysia

: Infection with Pantoea stewartii subspecies stewartii , which causes jackfruit-bronzing disease, is a huge problem faced by the jackfruit industry in Malaysia. This study was carried out to assess the disease ratings and aggressiveness performance of 28 Pantoea stewartii subspecies stewartii isolated from jackfruit-bronzing diseased jackfruits from four collection areas (Jenderam in Selangor State, Maran and Muadzam Shah in Pahang State, and Ipoh in Perak State) in Peninsular Malaysia, inoculated into jackfruit varieties (Tekam Yellow J33, Hong J34 and Subang Chap Boy J39), the sweetcorn variety, Mas Madu (two-week-old seedlings and nine-week-old seedlings), the cucumber variety, Rocky, and the pineapple variety, MD2. The results revealed that Pantoea stewartii subspecies stewartii produced symptoms upon all inoculations in the pathogenicity testing, thus fulﬁlling Koch’s postulates, except in the case of J39 and sweetcorn (two-week-old seedlings). No disease symptoms (disease rating 0) were observed in J39 and sweetcorn (two-week-old seedlings) within 14 days post-inoculation (14 dpi). The disease progression (based on disease ratings) proved that the jackfruit variety J39 was the most resistant, whereas J33 and J34 were susceptible to jackfruit-bronzing disease. The disease ratings of the 14-dpi period revealed a variability of disease progression among the 28 bacterial isolates, where the isolate JEN-14 had the fastest and highest disease ratings when inoculated into J33, J34, nine-week-old sweetcorn seedlings, cucumber, and pineapple. Likewise, the AUDPC value, based on disease rating, across the 28 isolates indicated that JEN-14 is the most aggressive and signiﬁcant of the isolates (J33, J34, nine-week-old sweetcorn seedlings, cucumber, and cucumber pineapple inoculation; p < 0.05). Even though isolates from Jenderam (other than JEN-14) and Maran had better disease ratings and the area under the disease progress curve (AUDPC) values than isolates from Muadzam Shah and Ipoh, no signiﬁcant differences were found among the isolates ( p < 0.05). Based on our ﬁndings, we identiﬁed the isolate JEN-14 as the best potential candidate to assist in jackfruit-bronzing disease resistance breeding in the future. Last but not least, the methods, disease ratings, and variations of the aggressiveness proﬁles among the isolates from this study may be beneﬁcial and signiﬁcant by providing disease-rating references and appropriate screening approaches when selecting the most appropriately aggressive isolates for evaluating the defense response in the disease resistance breeding program.

disease assessment, such as performing artificial inoculation using the most aggressive isolate, to evaluate the resistance of the various breed crop varieties [35][36][37][38]. In addition, the action of aggressiveness on different varieties or hosts heightens the disease progression and rating results as it often reflects the degree of host tolerance and susceptibility [39]. The assessment of disease performance, therefore, reflects the degree of host susceptibility and pathogen aggressiveness [39].
The results of previous pathogenicity testing of the jackfruit-bronzing disease reported the symptoms of P. stewartii subsp. stewartii-infected jackfruit, including reddish discoloration or bronzing, or rusty specks with yellowish discoloration of the jackfruit [7][8][9][10][11]. Moreover, the inoculation of sweetcorn and pineapple with P. stewartii subsp. stewartii elicited symptoms, causing the appearance of linear, pale yellow streaks with wavy margins. In addition, the fruit may turn brown and desiccate (Stewart's wilt symptom) in sweetcorn and trigger localized lesion symptoms in pineapple [10]. However, a study on the aggressiveness of the jackfruit-bronzing isolates via pathogenicity testing is still lacking. In addition, no pathogenicity test on different jackfruit varieties (other than the infected variety) has been conducted regarding the jackfruit-bronzing pathogen. Any study of the performance of the jackfruit-bronzing bacterium on resistance and susceptible jackfruit varieties is yet to be conducted. The only closely related study of pathogenicity tests for P. stewartii subsp. stewartii were the aggressiveness performance of P. stewartii subsp. stewartii isolates that were isolated from infected Zea mays (Stewart's wilt) in Indonesia on hybrid, open-pollinated and sweetcorn maize varieties by [40]. Their findings ascertained that sweetcorn varieties were the most susceptible, compared to the other two.
Therefore, the current study emphasizes the aggressiveness of the jackfruit-bronzing pathogen, based on pathogenicity tests, with the aims of (i) developing a disease rating reference guide (with pictures) and (ii) determining and assessing the aggressiveness of P. stewartii subsp. stewartii isolates when inoculated into jackfruit varieties, cucumber, and pineapple, using isolates collected from four sites in Peninsular Malaysia. Furthermore, the outcome from this study would assist in the breeding of jackfruit-bronzing-resistant varieties that may offer a similar taste and quality to J33.

Bacterial Isolates
Twenty-eight bronzing-related disease isolates (Table 1) from outbreaks in jackfruit plantation areas in Peninsular Malaysia (Jenderam from Selangor State, Maran and Muadzam Shah from Pahang State, and Ipoh from Perak State) were selected from a previous study [8]. All 28 isolates were verified as P. stewartii subsp. stewartii and as pathogenic (with a positive hypersensitivity reaction (HR)) [8]. The isolates were grown from a frozen glycerol stock onto nutrient broth yeast (NBY) and King's B media for 24-48 h at 28 • C for pathogenicity and inoculation purposes.

Pathogenicity Test
Different varieties of detached jackfruit J33, Hong (J34), and Subang Chap Boy (J39) were used in this experiment. These healthy jackfruit varieties were provided by the DOA, Malaysia. A pathogenicity test was performed with jackfruit flesh removed from disinfected healthy jackfruit, rinsed with a small amount of 10% ethanol, and washed twice with distilled water to ensure that there was no contamination. The jackfruit pulps were dried on sterile tissue paper and placed in a sterile petri dish with a layer of sterile filter paper. The pathogenicity test was conducted by injecting 10 mL of a 10 8 CFU/mL (as determined by McFarland Standard, bioMérieux, Marcy-l'Etoile, France) suspension of the pure P. stewartii subsp. stewartii into jackfruit flesh (4 replicates per isolate) [10]. The P. stewartii subsp. stewartii suspension was injected with a syringe to a depth of 10 mm, with 3 inoculation sites on each pulp. Control jackfruits were treated with sterile distilled water. The jackfruits inoculated with all 28 bacterial isolates or with the control (distilled water) were appropriately sealed using parafilm and incubated at 28 • C in a controlled chamber for the treatments. Observations and examinations for the development of symptoms were recorded daily for up to 2 weeks after inoculation. The assessment of bronzing symptoms was examined after 2 weeks of incubation. The experiment was repeated 3 times.  Pahang  2017  BSYD  MK802535  +  22  IPOH-5  AH  J33  Ipoh  Perak  2017  BSYD  MK531591  +  23  IPOH-B  AH  J33  Ipoh  Perak  2017  RD  MK802536  +  24  IPOH-I  AH  J33  Ipoh  Perak  2017  BSYD  MK802537  +  25  IPOH-M  AH  J33  Ipoh  Perak  2017  BSYD  MK802538  +  26  IPOH-S  AH  J33  Ipoh  Perak  2017  BSYD  MK802539  +  27  IPOH-V  AH  J33  Ipoh  Perak  2017  RD  MK802540  +  28  IPOH-Z  AH  J33  Ipoh  Perak  2017  RD  MK802541  + Abbreviations: NS 1 = no. of isolates; HR 2 = hypersensitivity reaction; AH 3 = Artocarpus heterophyllus; J33 4 = Tekam Yellow J33; RD 5 = reddish discoloration; BSYD 6 = rusty or bronzing specks with yellowish discoloration; + 7 = positive reaction.

Other Hosts Tests on Sweetcorn, Cucumber and Pineapple
Inoculation of P. stewartii subsp. stewartii was performed on the sweetcorn (Zea mays) variety, Mas Madu, the cucumber (Cucumis sativus) variety, Rocky, and the pineapple (Ananas comosus) variety, MD2. These healthy sweetcorn, cucumber, and pineapple fruits were provided by the DOA, Malaysia. A glasshouse experiment with P. stewartii subsp. stewartii on sweetcorn was performed on two-and nine-week-old healthy sweetcorn [10,23,41]. One milliliter of a 10 8 CFU/mL (as determined by McFarland Standard, BioMérieux, Marcy-l'Etoile, France) bacterial suspension was injected into the stem of the sweetcorn. The inoculation sites were covered with sterile tissue, parafilm, and duct tape. Three inoculation sites were used on each sweetcorn stem. All 28 bacterial isolate treatments, together with the control (distilled water), were performed with 4 replicates of sweetcorn. The treatments were randomized using a completely randomized layout with the statistical tool for agricultural research (STAR) software (http://bbi.irri.org/products, accessed on 10 May 2018). The glasshouse experiment was observed daily and was assessed 2 weeks after inoculation. The experiment was repeated 3 times. Fresh, healthy cucumbers and pineapple were cut into similar sizes (so as to fit inside the petri dish), rinsed with a small amount of 10% ethanol, and washed twice with distilled water to ensure that there was no contamination [10]. The cut fruit was dried on sterile tissue paper and placed on a sterile petri dish on a layer of sterile filter paper. Then, 1 mL of a 10 8 CFU/mL (as determined by McFarland Standard, BioMérieux, Marcyl'Etoile, France) suspension was injected into healthy cut pineapple and cucumber (4 replicates per isolate). Next, the bacterial suspension was injected with a syringe to a depth of 10 mm into the pineapple and 5 mm into the cucumber, with 3 inoculation sites on each cut fruit.
For the treatments, the pineapple and cucumber, inoculated with all 28 bacterial isolates and the control (distilled water), were appropriately sealed using parafilm and incubated at 28 • C in a controlled chamber. The experiment was repeated 3 times. The observations and examinations for the development of symptoms on the pineapple and cucumber were recorded daily for up to 2 weeks of incubation. The samples were assessed after 2 weeks of incubation. The experiment was repeated 3 times.

Disease Rating Assessment
Assessment for the pathogenicity test and other hosts' inoculation (except for Stewart's wilt symptom) was developed based on the symptoms obtained in the study. The disease rating score was rated (0-4) using (i) the RAL ® color chart [42], based on the color changes, and (ii) analysis using ImageJ software [43] and the Fiji distribution of ImageJ [43], based on the degree of severity of symptoms produced ( Figures S1 and S2). The observation of Stewart's wilt was based on a previous study on the loss of turgidity of the leaves or possibly of the entire plants [33]. Stewart's wilt symptoms on sweetcorn were scored according to an ordinal rating scale of 0-9, as illustrated by [44].

Assessment of AUDPC and Statistical Analysis
Data were presented as a median. Furthermore, the area under the disease progress curve (AUDPC) value of disease rating was calculated daily, from the initial day of the inoculation until the final day, using the following formula [45]: where n is the number of evaluations, y i is the values of the disease rating at the ith observation, and t i is the assessment period at the ith observation. Statistical analyses were conducted using R version v. 4.1.0. [46] and Rstudio v. 1.4.1717-3 [47], along with the R packages purrr, readxl, tidyverse, ggplot2, RColorBrewer, FSA, rcompanion, and patchwork [48][49][50][51][52][53][54]. The differences between data were subjected to the non-parametric Kruskal-Wallis test, followed by Dunn's post hoc test for the comparison of rank sums. The results were considered significant at p < 0.05.

Koch's Postulates on Jackfruit Varieties, Sweetcorn, Cucumber and Pineapple
After 14 days of inoculation, all symptomatic and asymptomatic inoculated jackfruit varieties, two-and nine-week-old sweetcorn, cucumber, and pineapple, were re-isolated to confirm the presence of the bacterium. The inoculated samples were put inside clear polyethylene bags (plastic surface sprayed with alcohol (70%)), brought under laminar flow for surface sterilization. Symptomatic and asymptomatic samples were excised, cut, and rinsed with sterilized distilled water (containing 1% of sodium hypochlorite, Chlorox™) for 3 min. The samples were then crushed and serially diluted 10-fold in sterile phosphate buffer (PB) [23]. Then, 100 µL of each dilution was spread over nutrient broth yeast (NBY) medium and King's B medium, appropriately sealed and incubated in an inverted position at 28 • C for 24 to 48 h. After incubation, isolated colonies were subcultured until pure colonies of suspected P. stewartii subsp. stewartii isolates were obtained. The expected colonies' morphology was lemon to orange-yellow or pale yellow, flat to convex, transparent with entire edges, and with medium growing. Biochemical tests consisted of Gram-staining, oxidase reaction, starch hydrolysis, gelatin liquefaction, motility test, and indole production were performed [10,16,18,23] as well as using a specific primer pair, ES16/ESIG2c [22], for the fulfillment of Koch's postulates.

Disease Rating Assessment on Jackfruit
An ordinal rating scale was developed, utilizing a reddish discoloration symptom scoring on jackfruits (Table 2 and Figure 1). Disease rating analysis for rusty or bronzing specks with yellowish discoloration symptoms was performed using ImageJ and the Fiji distribution of ImageJ. Rusty or bronzing specks with yellowish discoloration were developed and scored on an ordinal rating scale of symptoms, as illustrated (Table 3 and Figure 2). No disease symptoms appeared in the control jackfruit.

Disease Rating Assessment on Sweetcorn
Inoculation of sweetcorn led to symptoms of pale or yellowish-green leaf streaks on the nine-week-old seedlings, and no symptoms were observed on the two-week-old seedlings ( Figure 3). No disease symptoms appeared in the control seedling.   Table 3. The disease rating of rusty or bronzing specks with yellowish discoloration of jackfruit pulps (developed from this study).

Disease Rating Description
Rusty or bronzing specks with yellowish discoloration covered 1-24% of the jackfruit 2 Rusty or bronzing specks with yellowish discoloration covered 25-49% of the jackfruit 3 Rusty or bronzing specks with yellowish discoloration covered 50-74% of the jackfruit 4 Rusty or bronzing specks with yellowish discoloration covered > 75% of the jackfruit

Disease Rating Assessment on Sweetcorn
Inoculation of sweetcorn led to symptoms of pale or yellowish-green leaf streaks on the nine-week-old seedlings, and no symptoms were observed on the two-week-old seedlings ( Figure 3). No disease symptoms appeared in the control seedling.

Disease Rating Assessment on Cucumber
Reddish discoloration symptom scoring for cucumber was developed on an ordinal rating scale (Table 4 and Figure 4). No disease symptoms appeared in the control cucumber.

Disease Rating Assessment on Cucumber
Reddish discoloration symptom scoring for cucumber was developed on an ordinal rating scale (Table 4 and Figure 4). No disease symptoms appeared in the control cucumber.

Disease Rating Assessment on Pineapple
A disease rating analysis of the images taken was calculated, using ImageJ and the Fiji distribution of ImageJ. Localized lesion-symptom scoring on the pineapple was

Disease Rating Assessment on Pineapple
A disease rating analysis of the images taken was calculated, using ImageJ and the Fiji distribution of ImageJ. Localized lesion-symptom scoring on the pineapple was developed on an ordinal rating scale (Table 5 and Figure 5). No disease symptoms appeared in the control pineapple.
developed on an ordinal rating scale (Table 5 and Figure 5). No disease symptoms appeared in the control pineapple.

Disease Rating
Description 0 No symptom 1 Localized lesion area covered 1-24% of the pineapple 2 Localized lesion area covered 25-49% of the pineapple 3 Localized lesion area covered 50-74% of the pineapple 4 Localized lesion area covered > 75% of the pineapple   At four dpi, the majority of the jackfruit varieties J33 and J34 pulps, inoculated with the Jenderam and Maran isolates, developed symptoms (Figure 6a,b). They showed either reddish discoloration or rusty or bronzing specks with yellowish discoloration (Figures 1 and 2). The symptoms that appeared were similar to the appearance of the infected collected fruits (isolates from the reddish discoloration symptom samples induced a reddish discoloration symptom on the inoculated fruits, and isolates from rusty or bronzing specks with yellowish discoloration induced rusty or bronzing specks with yellowish discoloration on the inoculated fruits) ( Table S1). The appearance of symptoms on jackfruit varieties J33 and J34 inoculated with isolates from Muadzam Shah and Ipoh varied, manifesting at around eight to 14 dpi. No disease progression was observed on the MS-4-inoculated jackfruits for both varieties (Figure 6a,b). On the other hand, disease symptoms progressed faster in jackfruit varieties J33 and J34 when inoculated with the isolate JEN-14. No symptoms were observed on the isolated jackfruit variety J39 within 14 dpi (Figure 6c). Furthermore, no disease symptoms appeared in the control group of all jackfruit varieties.
The inoculated sweetcorn showed symptoms of pale or yellowish-green leaf streaks on the nine-week-old seedlings (Figure 3b,d and Figure 7a) but no symptoms showed on the two-week-old seedlings (Figures 3a and 7b). Like the jackfruit varieties J33 and J34, it took only four days for the nine-week-old seedlings to show Stewart's wilt symptoms when inoculated with isolates from Jenderam and Maran. In contrast, consistent results of symptom appearance were observed at seven and eight dpi when inoculated with the Muadzam Shah and Ipoh isolates (Figure 7a). In addition, the nine-week-old seedling that was inoculated with the isolate JEN-4 reached the maximum disease rating of 9 at 11-14 dpi, while the other isolates from Jenderam and Maran maximized at around a 3-8 rating at eight to 14 dpi, and isolates from Muadzam Shah and Ipoh maximized with a consistent rating of 2-3 at 11-14 dpi (Figure 7a). In contrast, the slowest and the lowest disease rating was in MS-4-inoculated seedlings (nine weeks old), only showing symptoms at 14 dpi with a disease rating of 1.
At seven and eight dpi, cucumber and pineapple inoculated with the Jenderam and Maran isolates showed symptoms of a reddish discoloration (Figures 4 and 8a) and localized lesions on the surface of the fruitlet (Figures 5 and 8b), respectively. The majority of the Muadzam Shah and Ipoh isolates elicited symptoms at 13 dpi in cucumber and at nine dpi in pineapple. Likewise, similar to J33, J34 and sweetcorn (nine weeks old) inoculation, the isolate JEN-14 still progressed faster than other isolates in cucumber and pineapple, whereas the isolate MS-4 was the lowest, with no disease progression (Figure 8a,b). No disease symptoms appeared in the control sweetcorn, cucumber, and pineapple.
The AUDPC value, based on the disease rating for J39 and two-week-old sweetcorn seedlings, was not performed due to no symptom being elicited after these inoculations (Figures 6c and 7b). The AUDPC value, based on the disease rating calculated from initial to 14 dpi on the inoculated jackfruit varieties (J33 and J34), sweetcorn (nine weeks old), cucumber, and pineapple, revealed that the isolate JEN-14 was the most aggressive by more than double, compared to other isolates (p < 0.05; Figure 9). The results illustrate how highly significant the isolate JEN-14 was compared with the rest of the isolates (p < 0.05; Figure 9). Despite the isolate MS-4 being observed as the least aggressive, based on the disease rating progression (Figure 6a,b, Figures 7a and 8a,b), not much significant difference was found between the isolate MS-4 and other isolates from Muadzam Shan, Ipoh, and several Jenderam isolates (Figure 9, p < 0.05). We can also see that the median values of isolates from Jenderam and Maran were around twice the median of isolates from Muadzam Shah and Ipoh (except for the isolate MS-4) (Figure 9). Therefore, it can be inferred that the isolate JEN-14 was more aggressive than the other 27 jackfruit-bronzing isolates collected around Peninsular Malaysia.

Koch's Postulates on Jackfruit Varieties, Sweetcorn, Cucumber and Pineapple
The isolation of the inoculated jackfruit varieties, sweetcorn, cucumber, and pineapple revealed P. stewartii subsp. stewartii, except on J39 (no symptom and no bacterial colony) and two-week-old seedlings (no symptom) (15 dpi). The spread-plate method of inoculated jackfruit varieties (except on J39), sweetcorn, cucumber, and pineapple produced bacteria colonies resembling P. stewartii subsp. stewartii morphology (lemon yellow color, Figure 10a) and were further subcultured; they showed positive P. stewartii subsp. stewartii identification (Figure 10b). The subcultured bacterial colonies were tested further using biochemical tests, which showed positive P. stewartii subsp. stewartii identification (Table S2) Figure 10f); a negative motility test was seen for all isolates as the inoculation line was still confined to the stab line. There were no color changes after Kovac's reagent was added to the SIM media, which remained yellow, and the indole production test was negative (Figure 10g). PCR amplification of bacterial colonies from all isolation of inoculated fruits/seedlings (except J39) each produced a 920 bp amplicon (Table S2 and

Discussion
Data from the pathogenicity tests on the jackfruit varieties showed the same characteristic symptoms of fruit bronzing for the jackfruit varieties J33 and J34. However, no symptom was observed for 14 days on variety J39. Based on the AUDPC value between the three varieties, J33 was the most susceptible, followed by J34, and J39 was the most resistant. Previous findings on these 28 isolates confirmed the presence of hrp (a virulence and pathogenicity gene) and a positive HR on tobacco of P. stewartii subsp. stewartii causing jackfruit-bronzing disease [8]. The type III secretion system (T3SS) protein and hrp gene are the main pathogenicity and virulence factors for P. stewartii subsp. stewartii [55][56][57], where this hrp gene encodes the T3SS and plays a vital role in plant pathogenic bacteria, eliciting disease in susceptible plants.
The authors of [58] have suggested that exopolysaccharides (EPSs) produced by plant pathogenic bacteria can avoid or delay the activation of plant defenses. However, when tested under in vitro conditions, many EPSs were produced and activated only when the culture medium of P. stewartii subsp. stewartii contained fermentable sugar, such as glucose and sucrose [59]. The results suggested that nutrient availability could regulate the expression and activation of the hrp gene in plant pathogens [60]. Thus, the nutrient availability of the host is necessary to activate the plant pathogen. Interaction of P. stewartii subsp. stewartii with a living host with high nutrients strengthen the pathogen growth level [59]. Furthermore, [61] reported that J33 and J34 varieties have sweet pulps with 27-30% brix and 11.6-14.2% brix, respectively, while J39 has less sweet to moderately sweet pulps, which proved the difference in disease progression.
For sweetcorn inoculation, our results revealed no disease symptoms in the two-weekold seedlings for 14 days (Figure 7b), even though the isolation at 15 dpi resulted in the positive presence and confirmation of P. stewartii subsp. stewartii (Table S2). We recommend using older age-stage seedlings as inoculation into young sweetcorn leaves may lead to a false symptom and disease assessment. This scenario was shown in the current study, where the nine-week-old seedlings produced a similar result, an atypical symptom of Stewart's disease, as demonstrated in maize and sweetcorn inoculated with Stewart's wilt pathogen [41,44] and jackfruit-bronzing pathogen [10]. Very young sweetcorn or maize is prone to no lesions or no development of other disease symptoms [41].
The results observed from the inoculation of pineapple showed a similar pattern to that demonstrated by [10], which produced localized rotting symptoms. Unfortunately, the results for the cucumber inoculation obtained from the experiment contradict a study by [10]. The researchers [10] did not observe any results from the inoculation, whereas this current study showed a reddish discoloration symptom similar to bronzing on jackfruit. The symptoms elicited in the cucumbers were similar to cucumber disease caused by Pseudomonas syringae pv. lachrymans, known as angular leaf spot, which causes soft rot and reddish-brown discoloration on and inside the cucumber fruit [62]. Pectobacterium carotovorum subsp. brasiliense, which causes bacterial soft rot in cucumber, also elicited similar symptoms of reddish-brown discoloration in the internal fruit part [63]. As mentioned previously, the T3SS protein and the hrp gene are the main pathogenicity and virulence factors for P. stewartii subsp. stewartii. Further study has confirmed that a T3SS protein of P. stewartii subsp. stewartii, known as WtsE, causes cell death activity and suppresses plant defenses against the non-host plants Arabidopsis thaliana, Nicotiana benthamiana and Beta vulgaris cv. Red Ace Hybrid (beet) [56,57].
Kruskal-Wallis analysis, followed by Dunn's post hoc test of the AUDPC, based on disease rating among isolates, confirmed that only the isolate JEN-14 was highly significant compared with the other 27 isolates. The consistency of the isolate JEN-14 as the most aggressive isolate when inoculated into J33, J34, sweetcorn (nine-week-old sweetcorn), cucumber, and pineapple, as well as the 0-rating disease progression on J39 (resistance variety), may be beneficial and applicable for future use in resistance screening and breeding against jackfruit-bronzing disease. Using the least aggressive isolate may lead to false disease breeding evaluation. It may show a similarly low rating when inoculated into susceptible and resistant varieties.
There were differences in the disease progression performance and the AUDPC based on disease ratings according to collection area. Isolates collected from Jenderam and Maran progressed better. They had a higher median than isolates collected from Maran and Ipoh, even though no significant correlation was found among the isolates (except for isolate . According to [64], variations in the pathogenicity and aggressiveness of plant pathogens could also arise due to the ecological environment and host diversity. Additionally, bacterial competence and genomic modifications are influenced by stressful environmental conditions and habitat adaptation [65]. Therefore, in further studies on factors besides aggressiveness that contribute to the outbreak of jackfruit-bronzing disease, factors such as soil and environmental conditions (i.e., temperature, relative humidity, and rainfall) should be considered for effective control of jackfruit-bronzing disease in Peninsular Malaysia.
This study documented for the first time the pathogenicity symptoms of P. stewartii subsp. stewartii isolates on jackfruit varieties, sweetcorn, cucumber, and pineapple, based on a disease rating assessment of jackfruit-bronzing disease from the four sites of isolate collection in Malaysia. Our study aids and guides using a pictorial disease-rating reference of P. stewartii subsp. stewartii pathogenicity on jackfruit and with the inoculation of cucumber and pineapple, based on visual and image-based assessments. Not only will these pictorial disease rating scales and assessments be valuable in the future for plant breeders but they can also benefit researchers, especially plant pathologists, as they provide more information on the diseases and guides to investigate further how far and how fast jackfruit-bronzing disease has progressed, thus allowing for the development of management control strategies to combat the spread of the disease.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/agronomy11112113/s1, Table S1: Symptoms of the isolates from the collected infected jackfruit (Artocarpus heterophyllus variety Tekam Yellow J33) from the four infected collection areas and symptoms induced on inoculated jackfruit pulps (n = 28); Table S2: Biochemical and molecular characterization of the isolates isolated from the inoculated jackfruit varieties, sweetcorn nine-weekold seedlings, cucumber and pineapple at 15 days post-inoculation; Figure S1: Image assessment for the area of infected pineapple fruitlet using ImageJ and Figure S2: Image assessment for bronzing and rusty specks with yellowish discoloration of infected jackfruit pulp using ImageJ.