A Mechanistic Study of Citrus Infection by Penicillium expansum

ZHAO Lina, LI Huifang, YU Jiang, LIU Kun, ZHANG Xiaoyun, YANG Qiya, ZHANG Hongyin*

(School of Food Science and Biological Engineering, Jiangsu University, Zhenjiang 212013, China)

Abstract: Strain H1, isolated from rotten citrus wounds in our laboratory, was identified as Penicillium expansum and was found to be able to produce citrinin (CIT). In this study, the possible mechanisms underlying citrus infection by P. expansum were investigated by measurement of cell wall degrading-enzymes and transcriptome analysis. The results showed that P.expansum H1 could produce polygalacturonase, pectin methylgalacturonase, endo-1,4-β-D-glucanase and β-glucosidase in both modified Marcus medium and infected citrus tissues. Meanwhile, the expression levels of all genes related to the infection were up-regulated, such as those related to cell wall degrading-enzymes, CIT production, the pH changes of the host, antioxidative stress response and effector factors. The results indicated that cell wall-degrading enzymes, CIT, pH, and antioxidant enzymes all play vital roles in citrus infection by P. expansum H1.

Keywords: Penicillium expansum; citrus; infection; enzyme activity; transcriptome

Citrus is one of the world’s major fruit crops with global availability and popularity contributing to human diets[1].China is the largest citrus-producing country around the world, while the loss from postharvest decay is considerable.Penicillium digitatum and Penicillium italicum are the most devastating pathogens of citrus, and they are responsible for important economiclosses during postharvest handling[2].P. expansum is also one of the pathogenic fungi in citrus postharvest[3]. Especially, P. expansum can produce secondary metabolites, such as patulin (PAT) and citrinin (CIT), which are harmful to human health[4]. Nowadays, while the use of synthetic fungicides can effectively control pathogens of postharvest fruits and vegetables as the primary strategy,there is an increased concern about the potential harmful effects on human health and environment of fungicide residues. In spite of the application of fungicides and the increased implementation of new alternative strategies(such as biological control)[5], green mold and blue mold decay in citrus fruits continue to impose high infection pressures on stored fruits worldwide. These facts justify the need and the interest for more detailed studies on hostpathogen interactions to increase our knowledge of virulence mechanisms of both pathogens and host defense mechanisms.This will serve as an initial step to fi nd out more rational of new and safer control strategies.

Approximately, all the described fungal species can cause diseases in plants. A common feature of this process is the necessity to pass through the plant cell wall, an important barrier against pathogen attack[6]. To this end, fungi possess a diverse array of secreted enzymes to depolymerize the main structural polysaccharide components of the plant cell wall, such as cellulose, hemicellulose, and pectin[7]. Recent advances in genomic and systems-level studies have begun to unravel this diversity and have pinpointed cell walldegrading enzyme (CWDE) families that are specifically present or enhanced in plant-pathogenic fungi[7]. Pectinases are a complex group of enzymes that degrade various pectic substances present in plant tissues and mainly include pectin methylesterase (PME), pectin methylgalacturonase (PMG),polygalacturonase (PG), pectinlyase (PNL), pectin methyl trans-eliminase (PMTE) and other lyase[8]. The substrates of PME, PG and PMG are pectin, pectic acid and pectin,respectively[9]. It has been reported that pectin hydrolase can cause the fruit softening and decay by degrading the pectin in the intercellular layer[10]. Also, treatment of host cell walls with PG can enhanced the expression levels of CWDEs, which has been confirmed to be closely related to pathogens infecting plants[11]. Cellulase degrades cellulose to glucose by hydrolyzing β-1,4-glucoside bond[12].According to the method of hydrolysis and the specificity of substrate, the cellulose can be divided into three major types: endo-1,4-β-D-glucanase (Cx), exo-1,4-β-glucanase(C1) and β-glucosidase (β-G).

Recent advances in genomics, transcriptomics and proteomics technologies make hunting for the research on interaction between plants and pathogens much more efficient. Transcriptome analysis can provide a comprehensive understanding of molecular mechanisms involved in specific biological processes and diseases from the information on gene structure and function.Transcriptome has been challenging due to the efficient and fast procedures of RNA-seq. RNA-seq, refers to the use of high-throughput sequencing technologies to sequence cDNA library transcribed from all RNAs in tissues or cells, can be used to quantify, profile, and discover RNA transcripts by sequence reads. Thus, the transcripts can then be mapped on the reference genome to get comprehensive genetic information. RNA-Seq has been widely used in biological,medical, clinical and pharmaceutical researches[13]. However,there have been only a few reports to explore the mechanism of pathogens infection on fruits.

In recent years, green mold decay of citrus caused by P. expansum is increasingly serious, P. expansum has a broad host range, such as apples[14], pears[15], peaches[16],tomatoes and citrus. However, there is no related report on the mechanism of P. expansum-infecting citrus at present.To understand the interaction between citrus fruits and pathogens is of great significance to find effective control strategies. Therefore, it is urgent to study the mechanism of P. expansum infection of citrus, which will provide a necessary theoretical basis for the prevention and control of green mold decay of citrus. In this paper, P. expansum H1, which was isolated by our laboratory from the rotten citrus, was chosen as the study subject. This study aimed to investigate the possible infection mechanisms of P. expansum infecting the citrus, from the aspects of cell wall-degrading enzymes and transcriptome technology. This study will reveal the possible infection mechanisms of P. expansum infecting the citrus, and will serve as an initial step to find out the rational of new and safer control strategies.

1 Materials and Methods

1.1 Material and reagents

P. expansum H1 was isolated from the rotten citrus by our laboratory, and we found that it can produce CIT[17]. The strain P. expansum H1 was incubated on potato dextrose agar medium (PDA medium, which contains 200 mL of extract from 200 g boiled potatoes, 20 g of dextrose, 20 g of agar and 800 mL distilled water) for 7 d at 25 ℃ before use. The concentration of P. expansum H1 was adjusted to 1 × 105spores/mL with sterile distilled water by using a hemocytometer.

Citrus (Citrus reticulata Blanco) were harvested at commercial maturity from an orchard in Zhenjiang, Jiangsu Province, China, and selected for uniformity of size, ripeness,and absence of apparent injury or infection for experiments.Fruits were selected randomly and sterilized with 0.1%sodium hypochlorite for 2 min, and washed with tap water in order to remove residual sodium hypochlorite, then air-dried at room temperature.

TRIzol was purchased from Tiangen Biotech (Beijing)Co. Ltd.. All the other reagentswere purchased from Sinopharm Chemical Reagent (Shanghai) Co. Ltd..

1.2 Instrument and equipment

ABI StepOnePlus Real-Time Polymerase Chain Reaction (PCR) Systemsand NanoDrop One were purchased from Thermo Fisher Scientific, USA; 2100 System Bioanalyzer was purchased from Agilent Technologies Inc., USA.

1.3 Methods

1.3.1 Determination of the lesion diameter of citrus infected by P. expansum H1

Three uniform wounds (3 mm diameter, 3 mm deep)were made at the equator of each citrus fruit using a sterile borer. 30 μL of suspension of P. expansum H1 at 1 × 105spores/mL was pipetted into each wound site, and sterile distilled water was used as control. 2 h later, the citrus samples were stored in enclosed plastic trays at 25 ℃ with 95% relative humidity (RH). The lesion diameter of citrus was measured by Vernier caliper after 3 d of incubation.

1.3.2 Determination of the activities of related cell wall degrading enzymes

1.3.2.1 The activities of related cell wall degrading enzymes in vitro

The strain P. expansum H1 was incubated on PDA medium by coating method for 7 d at 25 ℃ before use,and selected with the same growth conditions of mycelium by sterile borer (3 mm diameter), then transferred it to the inducible enzyme-producing medium of modified Marcus medium[18](Preliminary studies have shown that this medium allows the growth of the fungi and the production of a wide range of CWDEs, the medium containing VB10.2 mg, FeSO40.01g, MgSO4·7H2O 0.5 g, KCl 0.5 g, L-asparagine 0.5 g,KNO32 g, and K2HPO41.0 g, carboxy methyl cellulose sodium 10.0 g and 1 000 mL distilled water, adjust pH to 5.0)cultured at 120 r/min and 25 ℃ for 14 d. The samples were collected at 0, 2, 4, 6, 8, 10, 12, and 14 d for analyzing the related CWDEs in vitro. The fungal mycelia were removed by suction filtration before assay.

Sodium citrate buffer (50 mmol/L, pH 5.0) was used for the CWDEs’ assays. The activities of PG, PMG, Cx and β-G were assayed by the 3,5-dinitrosalicylic acid(DNS) modified method[19-20]. PG activity was assayed according to the method described by Douaiher[19], with 1%polygalacturonic acid as substrate at pH 5.0 and 50 ℃. PMG activity was measured by using 1% pectin as substrate at pH 5.0 and 50 ℃. The corresponding reducing sugar control was D-galacturonic acid. Cx and β-G activities were measured by using 1% carboxy methyl cellulose and 1% salicylin as substrate, respectively, at pH 5.0 and 50 ℃. For both Cx and β-G enzymes, the reducing sugar control was glucose. The reaction mixture (0.5 mL of crude enzyme solution, 1 mL of distilled water and 1.5 mL of substrate) for these CWDEs’assays was incubated for 30 min, then 3 mL of DNS was added to terminate the reaction. After boiling water bath for 5 min and cooled rapidly, distilled water was added to 20 mL.Absorbance was measured at 540 nm. A quantity of 1 U/mL is the amount of enzyme necessary to release 1 µg of reducing sugar/D-galacturonic acid min/mL of enzyme solution in the correspondent pH and temperature conditions.

1.3.2.2 The activities of related cell wall degrading enzymes of citrus (in vivo)

Citrus were prepared and wounded as described above.30 μL of suspension of P. expansum H1 at 1 × 105spores/mL was pipetted into each wound site, respectively, and sterile distilled water was used as control. 2 h later, the citrus samples were stored in enclosed plastic trays at 25 ℃ with 95% RH. In order to measure the activities of related cell wall-degrading enzymes, the tissue surrounding of each citrus was collected at 0, 2, 4, 6, 8, 10, and 14 d after infection.There were three replicates per treatment and the experiment was conducted twice. Five grams of tissue sample was ground with 20 mL of NaCl (1 mol/L) extracting solution, the homogenates were then centrifuged at 7 000 r/min for 15 min at 4 ℃ and the supernatants were assayed. All the extraction procedures were conducted at 4 ℃. The activities of PG,PMG, Cx and β-G were determined as described in vitro.

1.3.3 RNA extraction and transcriptome sequencing

Total RNA was isolated from 0.1 g sample with TRIzol.Citrus were prepared and wounded as described above. 30 μL of suspension of P. expansum H1 at 1 × 105spores/mL was pipetted into each wound site, and the citrus samples were stored in enclosed plastic trays at 25 ℃ with 95% RH for 3 d.P. expansumH1 infected citrus at the 3rdday, the mycelium ofP. expansumH1 was picked from the citrus wound and used as the experimental group, and marked as T3d, andP. expansumH1 (without infecting citrus, harvest from the liquid medium) as control, marked as T0d. The integrity of RNA was analyzed by using the RNA Nano 6000 Assay Kit of the Agilent Bio-analyzer 2100 system. The concentration and purity of RNA were measured by NanoDrop One.

Each RNA sample ofP. expansumH1 T3d and T0d was used to perform RNA-seq. Three micrograms of RNA sample was used for construct the cDNA libraries and sequencing.RNA sequencing libraries were constructed by using the NEBNext®Ultra RNA Library Prep Kit for Illumina with multiplexing primers. With non-stranded library preparation,the cDNA libraries were constructed by insert 150-250 bp,average 200 bp. Then an AMPure XP beads was used to purify the cDNA. After end repair and adapter ligation of the purified double cDNA chains, the appropriate fragments were screened by Agencourt AMPure XP beads, and the library was obtained by PCR enrichment. The library was sequenced on the Illumina HiSeq 2500 with the pair-end mode (done by Genepioneer Biotechnologies Company). Transcriptome data were separately assembled by Trinity, BLAST compared with Swissprot, Gene Ontology, Cluster of Orthologous Groups of proteins, and Kyoto Encyclopedia of Genes and Genomes database were used to the unigene sequences, and obtain the annotation information of Unigene. To calculate the gene expression levels, the method of FPKM was used. The differential expression levels of two samples were analyzed by DEGSeq (1.20.0).

1.3.4 Real time-quantitative PCR

One micrograms RNA ofP. expansumH1 was used to reverse with reverse transcription kit according to the instruction. The expression levels of interested genes were performed by real time-quantitative PCR (RT-qPCR)according to the method described by Zhao et al.[14]. The primers used for amplification were designed by Oligo7, and the primer sequences were shown in Table 1. The expression level of gene was normalized to the level of Actin, the data was calculated using 2−ΔΔCTmethod as described by Schmittgen and Livak[21].

Table 1 Primer sequences used for RT-qPCR amplification of differentially expressed genes

1.4 Statistical analyses

A statistical analysis of the data was carried out using SPSS 17.0. The mean values and the standard error of the mean were calculated from the data obtained from two independent experiments. The differences between the means of the test were evaluated by Student’st-test andP< 0.05 was considered as significantly different.

2 Results and Analysis

2.1 Effect of P. expansum H1 on the lesion diameter of citrus

In order to investigate the effect ofP. expansumH1 infection in citrus, the lesion diameter of citrus inoculated byP. expansumH1 was measured. As shown in Fig. 1,P. expansumH1 grew quickly in the wound of citrus, and we found that the mycelia were filled in the wound, however,when added the sterile distilled water in the wound of citrus,no mycelium was found. After 3 d, the lesion diameter in citrus inoculated byP. expansumH1 was 7.63 mm, and the control was 0 mm. Therefore,P. expansumH1 could infect the citrus and could cause decay quickly.

Fig. 1 Effect of P. expansum H1 on lesion diameter of citrus

2.2 The activities of the related cell wall degrading enzymes in vitro

As shown in Fig. 2, PG, PMG, Cx and β-G enzymes were produced by P. expansum H1 in modified Marcus medium. These activities of different cell wall-degrading enzymes had the same trend in the whole storage period, they all increased quickly, and reached a peak on day 6, except the activity of β-G enzyme which reached a peak on day 8,then began to decrease. On day 6, the activities of PG, PMG and Cx enzymes reached a peak, they were 11 440.03 U/mL,2 522.1 U/mL, and 168.9 U/mL, respectively. The activity of β-G enzyme was 153.3 U/mL on day 8.

Fig. 2 Activities of related cell wall-degrading enzymes of P. expansum H1 in vitro

2.3 The activities of related cell wall degrading enzymes of citrus (in vivo)

As shown in Fig. 3, the activities of PG, PMG, Cx and β-G enzymes of P. expansum H1 infecting citrus were investigated. PG activity in citrus treated with P. expansum H1 significantly increased, as compared with that in control,during the whole storage time (P < 0.05). On the 4thday, the activity of PG reached the fi rst peak (3 339.3 U/mL), which was 10.2-fold of the control, and PG activity reached the second peak (3 148.6 U/mL) on the 10thday. The activities of PMG, Cx and β-G were all significantly increased as compared with those of the control during the whole storage time. The activity of Cx reached the peak on the 6thday, was 318.6 U/mL, and then decreased, and β-G activity reached the peak on the 10thday, was 100.5 U/mL.

Fig. 3 Changes in related enzymes activities of citrus infected by P. expansum H1

2.4 Transcription and functional analysis of genes involved in P. expansum H1 infecting citrus

By sequencing, the large scale transcriptome data were obtained from P. expansum H1 (Table 2), there were a total of 31.72 Gb Clean data and high quality sequences, which were suitable for subsequent analysis.

Table 2 Statistic results of sequencing

Differentially expressed gene (DEG) are the genes differently expressed under two different conditions. In this study, the differently expressed unigenes ofP. expansumH1-infected citrus at the 3rdd and 0 d were analyzed. The results showed that a total of 230 differentially expressed genes(Fold change≥2 and FDR < 0.05) were identified, including 178 significantly up-regulated genes and 52 down-regulated genes.P. expansumH1 infecting citrus at 3rdd was marked as T3d, and the control was marked as T0d. “T0d _vs_ T3d” were used to categorize differentially expressed genes, henceforth upregulated genes had a higher expression level in sample group T3d than in sample group T0d, and viceversa.

In order to analyze the functions of these differentially expressed genes inP. expansumH1, the GO, COG annotations and classification were conducted (Fig. 4 & Fig. 5). As shown in Fig. 4, these differentially expressed genes were divided into three GO function categories, including cellular component, molecular function and biological process. From the results of the category of biological process, the highest category of differentially expressed unigenes were metabolic process, cellular process and single-organism process, and there were also differentially expressed genes related to the response to stimulus, reproduction, signaling, biological regulation, and so on. There were 7 kinds of differentially expressed genes in the category of molecular function, and the differentially significant expressed genes related to catalytic activity, binding and transporter activity. A total of 9 kinds of differentially expressed genes in the category of cellular component, and the highest category of differentially expressed genes were cell, cell part and membrane.

The COG annotations and classification of differentially expressed unigenes were shown in Fig. 5, there were more than 34 differentially expressed unigenes related to amino acid transport and metabolism, as well as chromatin structure and dynamics, accounting for the highest proportion, followed by the genes related to nuclear structure,carbohydrate transport and metabolism, transcription, and then cytoskeleton, defense mechanisms, nucleotide transport and metabolism, and etc.

Fig. 4 GO enrichment analysis of differentially expressed genes

Fig. 5 COG classifications of differentially expressed genes

The KEGG enrichment analysis of the differentially expressed unigenes was shown in Fig. 6, 25 DEGs were divided into 3 categories (cellular process, environmental information processing, and metabolism), which were further divided into 9 pathways. The categories of metabolism included 6 items, which were carbohydrate metabolism,amino acid metabolism, metabolism of other amino acids,lipid metabolism, metabolism of cofactors and vitamins,metabolism of terpenoids and polyketides, and the numbers of them contained DEGs, which were 12, 9, 5, 4, 4, 1,respectively. The expression levels of the majority of genes involved in carbohydrate metabolism, amino acid metabolism and lipid metabolism were changed after P. expansum H1 infected on the citruson the 3rdday, indicating that the basal metabolism of P. expansum H1 was responsive to the defense response of the infection on citrus.

Fig. 6 KEGG enrichment analysis of differentially expressed genes

2.5 Relative expression levels of genes involved in P. expansum H1 infecting citrus at different times

In order to verify the differences of these genes expressed in P. expansum H1-infected citrus at different times, the mRNA levels of 9 genes, which were differently expressed genes and associated with infection of citrus, were analyzed by RT-qPCR. As shown in Fig. 7, their expression levels in P. expansum H1-infected citrus on the 3rdday were obviously higher than that of infecting citrus on the 0 day.These results were in consistent with the results of transcriptome data analysis. All of the 9 genes verified were up-regulated,similar trend was observed in the transcriptome analysis. Among them, there were 4 genes related to cell wall-degrading enzymes,1 gene was related to the production of CIT, 1 gene was related to pH of the host, 2 genes were related to antioxidative stress response, 1 gene was associated with effector factor.

Fig. 7 RT-qPCR validation of differentially expressed genes and results of transcriptome

3 Discussion

In the process of invasion in plant cells, pathogenic fungi must penetrate the plant cell wall, an important barrier against pathogen attack[6]. To this end, fungi possess a diverse array of secreted enzymes to depolymerize the main structural polysaccharide components of the plant cell wall, such as cellulose, hemicellulose, and pectin[7]. Pectin polysaccharide is an important component of the adhesive layer in the plant cell wall, and acts as an adhesion of cell. When the adhesion is destroyed, the structure of plant tissue will be relaxed, which facilitates the further expanding of pathogens in plant[11].It has been reported that pectin hydrolase, such as PG and PMG, can cause the fruit softening and decay by degrading the pectin in the intercellular layer[10]. Cellulose is also one of the important components of plant cell wall, and it degrades eventually to glucose through hydrolyzing β-1,4-glucoside bond by cellulase[12], which includes Cx, C1 and β-G. In this study, the related CWDEs activities, including PG, PMG, Cx and β-G, were investigated in both modified Marcus medium(in vitro) and citrus disease tissues infected by P. expansum H1 (in vivo). The activities of PG, PMG, Cx and β-G in citrus infected by P. expansum H1 were all increased on day 3, as compared with those of citrus treated without P. expansum H1. It indicated that P. expansum H1 could infect citrus and colonized in it by destroying the cell wall through degrading pectinase and cellulase.

By the analysis of transcriptome data of P. expansum H1-infected the citrus, compared with citrus treated without P. expansum H1, a total of 230 differentially expressed genes were identified, including 178 significantly upregulated genes and 52 down-regulated genes. In order to verify the differences of genes expressed in P. expansum H1-infected the citrus, mRNA levels of 9 genes, which were differently up-regulated and related to infection. Among them, there were 4 genes related to cell wall-degrading enzymes. PEX2_013190, PEX2_110470, PEX2_056710 and PEX2_069880, encoding pectinesterase, PG, endo-1,4-βglucanase B and rhamnogalacturonase A, respectively, were all related to cell wall-degrading enzymes. Pectinesterase, PG and rhamnogalacturonase A belong to pectinase, and endo-1,4-β-glucanase B has the domain of cellulase. The expression levels of these four genes were all up-regulated, and related to cell wall-degrading enzymes, according with the increase of CWDEs activities in the citrus infected by P. expansum H1.It has been reported that Bcpg1 (encoding pectinase) is an important pathogenic factor in the process of Botrytis cinerea infecting Vitis vinifera[22]. Meanwhile, cellulase also plays an important role in the infection of pathogens. It indicated that P. expansum H1 infected citrus stimulated the expression levels of the genes encoding cell wall degrading enzymes,and cell wall-degrading enzymes is a pathogenic factor of P. expansum H1-infected citrus.

PEX2_044620 encodes polyketide synthase, which is involved in the synthesis of CIT[23]. The expression levels of PEX2_044620 were significantly up-regulated in the process of P. expansum H1 infecting citrus. It has been reported that the strain P. expansum H1 could produce CIT when it infected the citrus[17]. However, the role of CIT in the process of P. expansum H1 infecting citrus needs further research.

PEX2_038230 encodes a glucose-methanol-choline oxidoreductase, which is the precursor of glucose oxidase.Glucose oxidase can catalyze glucose to produce gluconic acid, which can change the pH of host. It has been found that P. expansum belong to “acid fungus”, which can induce the expression levels of the cell wall-degrading genes successfully in apples and other hosts through the acidification environment[24]. Our results showed that the expression levels of PEX2_038230 were up-regulated,indicating that the pH of citrus is lower when P. expansum H1 infects the citrus. This is consistent with previous studies[17].

PEX2_012540 encodes glutathione S-transferase,and PEX2_108950 encode catalase, they both belong to antioxidant enzymes, and are associated with antioxidant stress response. When pathogen infect the host, it will face the attack of the reactive oxygen species (ROS) produced by the infected part of the host, and the accumulation of ROS may cause damage to the pathogen cells[25-26]. Pathogens can develop several defense mechanisms to resist ROS,such as increase the defense of antioxidant enzymes and non-enzymatic protective molecules[27]. In this study, we found that the expression levels of PEX2_108950 were up-regulated, indicating that P. expansum H1 secrets antioxidant enzymes to resist oxidative stress of citrus.

PEX2_080220 encodes necrosis-inducing protein.When the host responds to the attack of the pathogen, which activates the systemic defense system, the different effector factors are secreted into the cytoplasm or cytoplasm. It not only induces the death of host directly, but also inhibits the defense response of the host, which is beneficial to the colonization of the pathogen[28-29]. Our results showed that the expression levels of PEX2_080220 was up-regulated,indicating that P. expansum H1 produces necrosis-inducing protein induced the death of citrus cells to facilitate its own infection.

In conclusion, the possible infection mechanisms of P. expansumon citrus through the aspects of cell wall degrading enzymes and transcription technology were investigated. From the aspects of cell wall degrading enzymes, the results showed that P. expansum H1 can produce PG, PMG, Cx and β-G in both modified Marcus medium and citrus disease tissues. Meanwhile, the differentially expression genes of P. expansum H1 after infecting citrus was compared and analyzed through transcriptome technology. It suggested that the expression levels of genes related to cell wall degrading enzymes, production of CIT, change in the pH of host, and also related to antioxidative stress response and effector factors were all up-regulated. Therefore, in the process of infection of citrus by P. expansum H1, P. expansum H1 may penetrate the cell wall of citrus through secreted cell wall-degrading enzymes, such as PG, PMG, Cx and β-G.Meanwhile, P. expansum H1 could produce CIT, and lower the pH of citrus, enhanced the antioxidative stress response,these factors are all beneficial to P. expansum H1 infection of citrus. The results indicated that cell wall-degrading enzymes,CIT, pH, antioxidant enzymes, are all the pathogenic effector in the process of P. expansum H1-infected the citrus, and the further infection mechanisms need to be understood.