Sugarcane Leaf Scald Research Articles

Plant Growth Promoting Microorganisms and Emerging Biotechnological Approaches for Sugarcane Disease Management

Sugarcane (Saccharum officinarum L.) is a highly valuable agricultural crop, cultivated globally in tropical and subtropical regions, primarily for its sugar content. Out of the 110 countries that grow sugarcane, India and Brazil together account for half of the world’s total production. Being an annual crop, it is prone to many diseases. The major diseases that can affect the sugarcane are red rot, wilt, sett rot, grassy shoot and pokkah boeng. Among the diseases Colletotrichum falcatum, Fusarium sacchari, Ceratocystis paradoxa, Candidatus Phytoplasma and Fusarium fujikuroi pathogen leads to more yield loss. Traditional disease management strategies, such as chemical treatments, conventional methods and biological control, offer limited protection throughout the crop cycle and raise concerns regarding environmental impact and sustainability. In recent years, plant growth-promoting microorganisms (PGPMs) have emerged as a promising alternative, offering environmentally friendly solutions to enhance plant health and manage diseases. RNAi has been explored to manage various diseases caused by viruses, fungi, and bacteria. By silencing key pathogenicity genes in the causal organisms, sugarcane mosaic virus (SCMV), smut, and leaf scald. The integration of PGPMs with RNAi emerging biotechnological tools and transcriptomics presents a sustainable approach to disease control, potentially reducing the dependency on chemical pesticides and promoting eco-friendly agricultural practices.

Transmission of Xanthomonas albilineans by the spittlebug, Mahanarva fimbriolata (Hemiptera: Cercopidae), in Brazil: first report of an insect vector for the causal agent of sugarcane leaf scald.

Leaf scald is a destructive sugarcane disease caused by the bacterium Xanthomonas albilineans (Ashby) Dowson. This pathogen presents the gene cluster SPI-1 T3SS, a conserved feature in pathogens vectored by animals. In this study, the competence of Mahanarva fimbriolata (Stål), a spittlebug commonly found in sugarcane fields in Brazil, was evaluated for the transmission of X.albilineans. Artificial probing assays were conducted to investigate the ability of M. fimbriolata adults to acquire X.albilineans from artificial diets containing the pathogen with subsequent inoculation of X.albilineans into pathogen-free diets. Plant probing assays with M. fimbriolata adults were conducted to evaluate the acquisition of X. albilineans from diseased source plants and subsequent inoculation of healthy recipient sugarcane plants. The presence of X.albilineans DNA in saliva/diet mixtures of the artificial probing assays and both insects and plants of the plant probing assays were checked using TaqMan assays. The artificial probing assays showed that M. fimbriolata adults were able to acquire and inoculate X.albilineans in diets. Plant probing assays confirmed the competence of M. fimbriolata to transmit X.albilineans to sugarcane. Over the entire experiment, 42% of the insects had acquired the pathogen and successful inoculation of the pathogen occurred in 18% of the recipient-susceptible sugarcane plants at 72 or 96 h of inoculation access period. Assays evidenced the vector competence of M. fimbriolata for transmission of X.albilineans, opening new pathways for investigating the biology and the economic impacts of the interaction between X.albilineans and M. fimbriolata.

In vitro and in vivo Antibacterial Activity of Three Natural Substances, for the Control of Xanthomonas albilineans Responsible for Sugarcane Leaf Scald in Côte d'Ivoire

In vitro and in vivo Antibacterial Activity of Three Natural Substances, for the Control of Xanthomonas albilineans Responsible for Sugarcane Leaf Scald in Côte d'Ivoire

Synergistic Effects of Organosilicon and Cu(OH)2 in Controlling Sugarcane Leaf Scald Disease.

Sugarcane leaf scald is a systemic disease caused by Xanthomonas albilineans that limits sugarcane yield and quality. Previous research has shown that exogenous application of copper hydroxide to plants is effective in controlling this disease. However, long-term bactericide use causes serious "3R" problems: resistance, resurgence, and residue. It is therefore urgent to discover new methods for the improvement of bactericide efficiency and efficacy. In the present study, disease index values for leaf scald were measured in sugarcane seedlings over time to determine the effects of different concentrations of copper hydroxide, types of silicon additive, and treatment timing after inoculation with X. albilineans on controlling sugarcane leaf scald disease. Our results show copper hydroxide mixed with organosilicon additive could improve the bactericide efficiency and efficacy and reduce the growth of pathogenic bacteria, even at a reduced concentration in both laboratory and field conditions. This study provides an important practical model for controlling sugarcane leaf scald disease by reducing the concentration of bactericide and increasing its efficacy in sugarcane fields.

Distribution, phylogeny, and pathogenicity of Xanthomonas albilineans causing sugarcane leaf scald in Mexico

Disease outbreaks attributed to leaf scald of sugarcane caused by Xanthomonas albilineans were recently reported in southern Mexico. In this study, the distribution of leaf scald was investigated in the main sugarcane producing-states in Mexico. The disease was found in eight of 14 sugarcane-growing states and in 12 of 31 fields. In contaminated fields, incidence of leaf scald based on symptomatic plants ranged from 3 to 15%. The causal agent was identified by phylogenetic reconstruction using a single concatenated sequence of four genes (16S rRNA, fusA, leuS, and rpiA). Twelve X. albilineans isolates from different locations in Mexico produced disease symptoms after inoculation into stalks of susceptible variety Mex 69–290. Microscopic examination of infected stalks revealed structural changes within vascular and non-vascular tissues. These alterations reflected acute disease development and highlighted the importance of selecting varieties resistant to X. albilineans in breeding programs in Mexico.

Naked eye evaluation and quantitative detection of the sugarcane leaf scald pathogen,

Sugarcane leaf scald caused by the bacterium Xanthomonas albilineans is a major disease of sugarcane worldwide. Whereas erratic symptoms make phenotypic detection challenging, molecular methods require expensive instruments and labour, and longer sample-to-answer times. We report a novel method for detection of X. albilineans DNA in sugarcane xylem sap. The method involves (i) boiling lysis-based DNA extraction from sugarcane sap; (ii) magnetic purification of target sequences directly from the lysate through use of magnetic bead-bound capture probes; and (iii) DNA sandwich hybridisation platform for HRP/TMB/H2O2 reaction-based naked eye visualisation and electrochemical detection of the target. The method is sensitive (limit of detection 100 fM) and reproducible (relative standard deviation <7%) with linear dynamic range 100 fM–1 nM (R2 = 0.99). The method was tested on a range of sugarcane cultivars of known resistance ratings (susceptible, intermediate resistant, and resistant) for leaf scald disease from an inoculated field trial. Detection levels agreed with the resistance rating of cultivars tested. In addition, qPCR results strongly correlated with our assay (r = 0.91–0.99, P < 0.01) and cultivar resistance rating. We believe that our assay could be useful for rapid screening as well as sensitive quantification of target pathogen DNA in infected sugarcane plants.

Identification and Phylogenetic Analysis of Xanthomonas albilineans (Ashby) Dowson Based on Multiple Gene Sequences in Yunnan Province, China

Sugarcane leaf scald caused by Xanthomonas albilineans (Ashby) Dowson is an extremely destructive disease. In 2017, suspected symptoms of sugarcane leaf scald was observed, in Baoshan, Mengding and Jinping, Yunnan Province, China. The bacteria was isolated from the symptomatic stalks and was subsequently identified as the causal agent using Koch’s postulates. BLASTN analysis showed that the amplified nucleotide sequences of 16S rRNA, gyrB and rpoD genes from all isolates shared 100% sequence identity with the nucleotide sequences of corresponding genes of X. albilineans strain GPE PC73 (Acc no. FP565176). Based on symptom diagnosis in the field, colony morphology, validation of Koch’s postulates and molecular identification of 16S rRNA, gyrB and rpoD genes, the disease was confirmed as sugarcane leaf scald caused by X. albilineans. Partial 16S rRNA, gyrB and rpoD gene sequence analysis of 36 X. albilineans strains from Yunnan and Guangxi provinces, China, was determined. The results revealed that the gyrB gene was the most sensitive in differentiating X. albilineans strains from Yunnan and Guangxi provinces.

Comparison of loop-mediated isothermal amplification, polymerase chain reaction, and selective isolation assays for detection of Xanthomonas albilineans from sugarcane

A loop-mediated isothermal amplification (LAMP) assay was developed and compared to polymerase chain reaction (PCR), nested PCR, and selective isolation assays for detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald. The pathogen was isolated on selective medium from 44 out of 45 (98%) samples taken from symptomatic stalks, and from 44 out of 70 (63%) samples from asymptomatic stalks that were collected from plots with symptomatic stalks. Forty-two (93%), 41 (91%), and 42 (93%) symptomatic samples tested positive by LAMP, PCR and nested PCR, respectively. The pathogen was detected in 19 (27%), 8 (11%), and 25 (36%) of the 70 asymptomatic samples by LAMP, PCR and nested PCR, respectively. Symptomatic stalks were mainly, but not always, associated with high populations of the pathogen (107–109 CFU/ml), and asymptomatic stalks with low populations (<103 CFU/ml) or no bacteria. Although our LAMP and nested PCR methods detected 10 CFU/ml of X. albilineans in suspensions prepared with pure culture, they sometimes failed to detect the pathogen in samples with low pathogen populations. Isolation on selective medium along with another method should therefore be used for detection of the pathogen in asymptomatic stalks, especially in quarantine programs.

Molecular detection and prevalence of Xanthomonas albilineans, the causal agent of sugarcane leaf scald, in China

Leaf scald caused by Xanthomonas albilineans (Ashby) Downson is a destructive disease of sugarcane that can cause severe economic losses. During 2013–2017, 357 sugarcane leaf samples were collected from four provinces in China: 141 samples were taken from plants showing symptoms attributed to leaf scald and 216 were taken from asymptomatic plants. A previously described PCR protocol and PCR primers (XAF1/XAR1) targeting a putative ATP (adenosine triphosphate)-biding cassette (ABC) transporter gene (XALc_1791) were used to detect X. albilineans in these leaf samples. The pathogen was detected in 73% and 17% of symptomatic and asymptomatic leaf samples, respectively. The PCR-amplified fragments (608 bp) from 140 Xa-positive samples were cloned and sequenced. A total of 241 XALc gene sequences were obtained and compared to 13 sequences of X. albilineans isolates retrieved from GenBank. The isolates from China had 99.1–100% sequence identity with one another and 99.5–100% with four strains from the French West Indies (GPE PC73, GPE PC17, GPE PC86, and MTQ032) and one from Florida/USA (XaFLO7-1). Our findings show that X. albilineans causes leaf scald of sugarcane in Guangxi, Guangdong, Hainan, and Fujian provinces, suggesting that this disease could be a potential threat to the sugarcane industry in China.

First Report of Sugarcane Leaf Scald Caused by Xanthomonas albilineans in the Province of Guangxi, China

Leaf scald caused by Xanthomonas albilineans (Ashby) Dowson is a destructive disease of sugarcane, as it causes severe economic losses (Rott et al. 1995). X. albilineans has been reported in over 60 countries in Africa, Asia, and North and South Americas (Rott et al. 2000), but has so far not been reported in mainland China. X. albilineans is a quarantine pathogen for sugarcane germplasm imported into China. In June 2016, symptoms of leaf scald were observed on the newly-bred sugarcane cultivars ‘Guitang46’ and ‘Guitang06-2081’ in Beihai, Laibin and Baise, Guangxi province, China. The average disease incidence of cultivars ‘Guitang46’ and ‘Guitang06-2081’ was 44.6% and 50.1%, respectively. Typical symptoms of the disease were white stripes along veins. Severely infected plants also exhibited shorter stalk internodes, smaller leaves, and abnormal development of side shoots along the stalk. Leaves of side shoots had similar white stripes as those observed at the top of the stalk. A sudden wilting of all lea...

Bioassay to Study the Attachment of Xanthomonas albilineans on Sugarcane Leaves.

Sugarcane (interspecific hybrids of Saccharum species) is an economically important crop that provides 70% of raw table sugar production worldwide and contributes, in some countries, to bioethanol and electricity production. Leaf scald, caused by the bacterial plant pathogen Xanthomonas albilineans, is one of the major diseases of sugarcane. Dissemination of X. albilineans is mainly ensured by contaminated harvesting tools and infected stalk cuttings. However, some strains of this pathogen are transmitted by aerial means and are able to survive as epiphytes on the sugarcane phyllosphere before entering the leaves and causing disease. Here we present a protocol to estimate the capacity of attachment of X. albilineans to sugarcane leaves. Tissue-cultured sugarcane plantlets were immersed in a bacterial suspension of X. albilineans and leaf attachment of X. albilineans was determined by two methods: leaf imprinting (semi-quantitative method) and leaf washing/homogenization (quantitative method). These methods are important tools for evaluating pathogenicity of strains/mutants of the sugarcane leaf scald pathogen.

Surface polysaccharides and quorum sensing are involved in the attachment and survival of Xanthomonas albilineans on sugarcane leaves.

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue-cultured plantlets grown in vitro. Six mutants of strain XaFL07-1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly-β-hydroxybutyrate than the wild-type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non-ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild-type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves.

Breaking dogmas: the plant vascular pathogen Xanthomonas albilineans is able to invade non-vascular tissues despite its reduced genome.

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is missing the Hrp type III secretion system that is used by many Gram-negative bacteria to colonize their host. Until now, this pathogen was considered as strictly limited to the xylem of sugarcane. We used confocal laser scanning microscopy, immunocytochemistry and transmission electron microscopy (TEM) to investigate the localization of X. albilineans in diseased sugarcane. Sugarcane plants were inoculated with strains of the pathogen labelled with a green fluorescent protein. Confocal microscopy observations of symptomatic leaves confirmed the presence of the pathogen in the protoxylem and metaxylem; however, X. albilineans was also observed in phloem, parenchyma and bulliform cells of the infected leaves. Similarly, vascular bundles of infected sugarcane stalks were invaded by X. albilineans. Surprisingly, the pathogen was also observed in apparently intact storage cells of the stalk and in intercellular spaces between these cells. Most of these observations made by confocal microscopy were confirmed by TEM. The pathogen exits the xylem following cell wall and middle lamellae degradation, thus creating openings to reach parenchyma cells. This is the first description of a plant pathogenic vascular bacterium invading apparently intact non-vascular plant tissues and multiplying in parenchyma cells.

Xanthomonas albilineansOmpA1 Appears to be Functionally Modular and Both the OMC and C-like Domains Are Necessary for Leaf Scald Disease of Sugarcane

Several EZ-Tn5 insertions in gene locus XALc_0557 (OmpA1) of the sugarcane leaf scald pathogen Xanthomonas albilineans XaFL07-1 were previously found to strongly affect pathogenicity and endophytic stalk colonization. XALc_0557 has a predicted OmpA N-terminal outer membrane channel (OMC) domain and an OmpA C-like domain. Further analysis of mutant M468, with an EZ-Tn5 insertion in the upstream OMC domain coding region, revealed impaired epiphytic and endophytic leaf survival, impaired resistance to sodium dodecyl sulfate (SDS), structural defects in the outer membrane (OM), and hyperproduction of OM vesicles. Cloned full-length XALc_0557 complemented M468 for all phenotypes tested, including pathogenicity, resistance to SDS, and ability to survive both endophytically and epiphytically. Another construct, pCT47.3, which expressed only the C-like domain of XALc_0557, restored resistance to SDS in M468 but failed to complement any other mutant phenotype, indicating that the C-like domain functioned independently of the OMC domain to help maintain OM integrity. pCT47.3 also complemented pathogenicity, resistance to SDS, and stalk colonization in mutant M1152, which carries an EZ-Tn5 insert in the C-like coding region, indicating that both predicted domains are modular and necessary but neither is sufficient for X. albilineans pathogenicity, endophytic survival in, and epiphytic survival on sugarcane.

First Report of Sugarcane Leaf Scald in Gabon Caused by a Highly Virulent and Aggressive Strain of Xanthomonas albilineans.

During a disease inspection at the sugarcane estate SUCAF near Franceville, Gabon, in March 2011, 0.5 to 1 cm wide chlorotic stripes covered with many small red streaks were observed on sugarcane (Saccharum spp.) leaves of a single plant of cultivar R581. After removal of the leaves covering the base of the stalks, abnormal development of basal side shoots was also observed. Transversal sections of a diseased stalk showed reddening of the vessels near the nodes. Circular, convex, smooth, shiny, translucent, non-mucoid, honey-yellow pigmented bacterial colonies were isolated from stalk pieces and side shoots on XAS selective agar medium (1). The nucleotide sequence of the 16S-23S internal transcribed spacer (ITS) of a representative colony was shown to be 100% identical to the 16S-23S ITS sequence from the genome of Xanthomonas albilineans strain GPE PC73 (GenBank: FP565176.1). This strain from Gabon was named GAB266. Sugarcane stalks of greenhouse grown cultivar CP68-1026 were inoculated with X. albilineans strains XaFL07-1 from Florida, GPE PC73 from Guadeloupe, and GAB266. Five stalks were inoculated by the modified decapitation method (3) with each strain or with a water control. One month post-inoculation (MPI), white pencil lines and severe necrosis were observed on leaves inoculated with strains XaFL07-1 and GPE PC73, and no disease symptoms appeared on non-inoculated leaves that developed 2 to 3 MPI. These results are in agreement with those generally obtained after inoculation of susceptible sugarcane cultivars with X. albilineans strains from various geographical locations under greenhouse conditions (Rott, unpublished results). In contrast, 1 MPI, only discrete white to red pencil lines were observed along with necrosis on leaves inoculated with strain GAB266, and by 2 to 3 MPI, all five inoculated plants were wilted. The pathogen was successfully reisolated by the stalk blot inoculation technique (3) with XAS medium, from all five inoculated stalks and from 98 of 114 internodes. In a second greenhouse experiment, the same three strains of X. albilineans were inoculated as described above into five sugarcane cultivars differing in resistance to leaf scald in Guadeloupe (2) (CP68-1026, highly susceptible; B69566, susceptible; R570, tolerant; B8008, resistant; Co6415, highly resistant). The same symptoms as those described above were again observed on inoculated leaves of the five sugarcane cultivars 1 MPI. Strains XaFL07-1 and GPE PC73 produced occasionally a single pencil line on non-inoculated leaves 2 to 3 MPI, but only strain GAB266 caused leaf scalding and/or plant death 2 to 3 MPI: cultivar CP68-1026 (5 of 5 plants), B69566 (5 of 5 plants), R570 (4 of 5 plants), B8008 (5 of 5 plants), and only non-inoculated leaves of cultivar Co6415 remained symptomless (5 plants). Strain GAB266 from Gabon appeared, therefore, more virulent and aggressive than the two strains of X. albilineans from Florida and Guadeloupe. To our knowledge, this is the first report of leaf scald of sugarcane in Gabon and the first description of an unusual highly virulent and aggressive strain of X. albilineans. A large-scale survey needs to be undertaken to determine the distribution of leaf scald disease and this new pathotype/race of X. albilineans in Gabon and other geographical locations.

Identification of New Candidate Pathogenicity Factors in the Xylem-Invading Pathogen Xanthomonas albilineans by Transposon Mutagenesis

Xanthomonas albilineans is a xylem-invading pathogen that produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms of sugarcane leaf scald. In contrast to other xanthomonads, X. albilineans does not possess a hypersensitive response and pathogenicity type III secretion system and does not produce xanthan gum. Albicidin is the only previously known pathogenicity factor in X. albilineans, yet albicidin-deficient mutant strains are still able to efficiently colonize sugarcane. To identify additional host adaptation or pathogenicity factors, sugarcane 'CP80-1743' was inoculated with 1,216 independently derived Tn5 insertions in X. albilineans XaFL07-1 from Florida. Sixty-one Tn5 mutants were affected in development of leaf symptoms or in stalk colonization. The Tn5 insertion sites of these mutants were determined and the interrupted genes were identified using the recently available genomic DNA sequence of X. albilineans GPE PC73 from Guadeloupe. Several pathogenicity-related loci that were not previously reported in Xanthomonas spp. were identified, including loci encoding hypothetical proteins, a membrane fusion protein conferring resistance to novobiocin, transport proteins, TonB-dependent outer-membrane transporters, and an OmpA family outer-membrane protein.

Genomic and Evolutionary Features of the SPI-1 Type III Secretion System That Is Present inXanthomonas albilineansbut Is Not Essential for Xylem Colonization and Symptom Development of Sugarcane Leaf Scald

Xanthomonas albilineans is the causal agent of sugarcane leaf scald. Interestingly, this bacterium, which is not known to be insect or animal associated, possesses a type III secretion system (T3SS) belonging to the injectisome family Salmonella pathogenicity island 1 (SPI-1). The T3SS SPI-1 of X. albilineans shares only low similarity with other available T3SS SPI-1 sequences. Screening of a collection of 128 plant-pathogenic bacteria revealed that this T3SS SPI-1 is present in only two species of Xanthomonas: X. albilineans and X. axonopodis pv. phaseoli. Inoculation of sugarcane with knockout mutants showed that this system is not required by X. albilineans to spread within xylem vessels and to cause disease symptoms. This result was confirmed by the absence of this T3SS SPI-1 in an X. albilineans strain isolated from diseased sugarcane. To investigate the importance of the T3SS SPI-1 during the life cycle of X. albilineans, we analyzed T3SS SPI-1 sequences from 11 strains spanning the genetic diversity of this species. No nonsense mutations or frameshifting indels were observed in any of these strains, suggesting that the T3SS SPI-1 system is maintained within the species X. albilineans. Evolutionary features of T3SS SPI-1 based on phylogenetic, recombination, and selection analyses are discussed in the context of the possible functional importance of T3SS SPI-1 in the ecology of X. albilineans.

Abstracts Submitted for Presentation at the 2010 APS Annual Meeting

Xanthomonas albilineans (Xa) is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Xa produces albicidin, a potent antibiotic and phytotoxin which blocks chloroplast differentiation, thus causing the foliar symptoms of the disease. Albicidin is the only known pathogenicity factor in Xa, yet albicidin deficient mutants are still able to colonize the sugarcane plant. In an attempt to identify other major pathogenicity factors, we screened 1,216 independent Tn5 insertions in Xa strain XaFL07-1 by single inoculation onto sugarcane cultivar CP80-1743. Mutants were screened for reduced pathogenicity (i.e., capacity to induce leaf symptoms and to multiply in the sugarcane stalk). Five independent Tn5 insertions were found in gene XALc_0557, which is predicted to encode an OmpA family outer membrane protein. Each of these insertions resulted in a mutant strain that elicited very slight to no symptoms and was not able to move as efficiently within the sugarcane stalk, both spatially and in intensity, as wild type XaFL07-1. Additional phenotypic studies showed that these mutants: 1) produced albicidin, 2) were less motile and 3) were slower growing than the wild type Xa in vitro. However, these OmpA mutants were able to multiply in sugarcane leaf tissue to levels similar to the wild-type strain XaFL07-1. Complementation analyses are currently underway. (Texte integral)

Epiphytic Populations of Xanthomonas albilineans and Subsequent Sugarcane Stalk Infection Are Linked to Rainfall in Guadeloupe.

Three separate field trials were established in Guadeloupe under different agronomic and rainfall conditions to study phyllosphere contamination and infection of sugarcane plants by Xanthomonas albilineans, the causal agent of sugarcane leaf scald. Disease-free and leaf scald susceptible cv. B69566 was planted and monitored during three 1-year crop cycles. Presence of leaf scald contaminated sugarcane fields in the proximity of the disease-free trials appeared critical in early contamination of the sugarcane phyllosphere. Later on, particular meteorological events, such as tropical storms, were also important in aerial spread of the pathogen. A positive correlation was found between epiphytic populations of X. albilineans and severity of leaf necrotic symptoms, but occurrence of leaf symptoms was not always related to subsequent stalk infection. However, when the data of the three crop seasons were considered together, a high correlation was found between rainfall and maximum epiphytic populations of X. albilineans, and between rainfall and subsequent stalk infections. Consequently, rainfall is a key factor to be considered in evaluation of risks of leaf scald epidemics, and protocols for propagation of healthy sugarcane material and screening methods for leaf scald resistance may have to be revised in humid tropical locations.

Substrate Specificity-Conferring Regions of the Nonribosomal Peptide Synthetase Adenylation Domains Involved in Albicidin Pathotoxin Biosynthesis Are Highly Conserved within the Species Xanthomonas albilineans

Albicidin is a pathotoxin produced by Xanthomonas albilineans, a xylem-invading pathogen that causes leaf scald disease of sugarcane. Albicidin is synthesized by a nonribosomal pathway via modular polyketide synthase and nonribosomal peptide synthetase (NRPS) megasynthases, and NRPS adenylation (A) domains are responsible for the recognition and activation of specific amino acid substrates. DNA fragments (0.5 kb) encoding the regions responsible for the substrate specificities of six albicidin NRPS A domains from 16 strains of X. albilineans representing the known diversity of this pathogen were amplified and sequenced. Polymorphism analysis of these DNA fragments at different levels (DNA, protein, and NRPS signature) showed that these pathogenicity loci were highly conserved. The conservation of these loci most likely reflects purifying selective pressure, as revealed by a comparison with the variability of nucleotide and amino acid sequences of two housekeeping genes (atpD and efp) of X. albilineans. Nevertheless, the 16 strains of X. albilineans were differentiated into several groups by a phylogenetic analysis of the nucleotide sequences corresponding to the NRPS A domains. One of these groups was representative of the genetic diversity previously found within the pathogen by random fragment length polymorphism and amplified fragment length polymorphism analyses. This group, which differed by three single synonymous nucleotide mutations, contained only four strains of X. albilineans that were all involved in outbreaks of sugarcane leaf scald. The amount of albicidin produced in vitro in agar and liquid media varied among the 16 strains of X. albilineans. However, no relationship among the amount of albicidin produced in vitro and the pathotypes and genetic diversity of the pathogen was found. The NRPS loci contributing to the synthesis of the primary structure of albicidin apparently are not involved in the observed pathogenicity differences among strains of X. albilineans.