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Diagnostic GuideOpen Access icon OPENOpen Access license

A Diagnostic Guide for Orange Rust Disease in Sugarcane

    Affiliations
    Authors and Affiliations
    • Daniela E. Cárdenas1
    • Yisel Carrillo-Tarazona1
    • Sushma G. Sood2
    • Martha A. Hincapie3
    • Jianping Wang4
    • Philippe C. Rott5 6
    • Liliana M. Cano1 †
    1. 1Department of Plant Pathology, Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Fort Pierce, FL 34945, U.S.A.
    2. 2Sugarcane Field Station, United States Department of Agriculture, Agricultural Research Service, Canal Point, FL 33438, U.S.A.
    3. 3Department of Plant Pathology, Everglades Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Belle Glade, FL 33430, U.S.A.
    4. 4Department of Agronomy, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, U.S.A.
    5. 5CIRAD, UMR PHIM, 34398 Montpellier, France
    6. 6PHIM, Plant Health Institute, Univ. Montpellier, CIRAD, INRAE, Institut Agro, IRD, Montpellier, France
    Published Online:17 Oct 2024https://doi.org/10.1094/PHP-02-24-0013-DG

    Abstract

    Puccinia kuehnii is an important airborne fungal pathogen of sugarcane, causing orange rust disease. P. kuehnii has been present in the Asia–Oceania region for over a century. The first report of this pathogen in the Western Hemisphere was in 2007, when it appeared in Florida, U.S.A. The main objective of this diagnostic guide is to provide detailed information about symptoms and signs of sugarcane orange rust as well as taxonomy, geographic distribution, isolation, identification, and storage of P. kuehnii. We also highlight the management strategies used in recent years to control this devastating fungal disease. This diagnostic guide will help facilitate future research focusing on fungal isolation procedures, genetics, and population studies of this pathogen.

    Hosts: Sugarcane (Saccharum interspecific hybrids) is affected by three different rust fungi worldwide (Puccinia melanocephala, P. kuehnii, and Macruropyxis fulva) (Rutherford 2018). Besides Saccharum species (S. officinarum, S. spontaneum, etc.) and Saccharum hybrids, P. kuehnii has been recorded to infect sugarcane-related species such as Erianthus arundinaceus, a Narenga sp., and Sclerostachya fuscum, also known as Miscanthus fuscus (Ryan and Egan 1989).

    Disease: Orange rust is the common name of the disease caused by P. kuehnii.

    Pathogen: P. kuehnii (W. Krüger) E. J. Butler (1919). P. kuehnii is a foliar biotrophic pathogen that infects the sugarcane leaf tissue, and the common name of the disease in sugarcane is orange rust (Chona and Munjal 1950). P. kuehnii was originally described as Uromyces kuehnii by Krüger in 1912 and as Uredo kuehnii as a synonym of this species (Butler 1919). Later on, this pathogen was reclassified based on its teliospores as P. kuehnii (Chona and Munjal 1950; Egan 1964).

    Taxonomy

    Kingdom Fungi, Phylum Basidiomycota, Subphylum Pucciniomycotina, Class Pucciniomycetes, Order Pucciniales, Suborder Uredinineae, Family Pucciniaceae, Genus Puccinia, Species P. kuehnii. The current taxonomic description can be found at MycoBank under accession 243512 (https://www.mycobank.org/page/Name%20details%20page/222667) and in the CABI Compendium (https://doi.org/10.1079/cabicompendium.45818).

    Symptoms and Signs

    In Florida, epidemics of P. kuehnii occur during the spring and summer seasons. The pathogen requires high humidity (>95%) and relatively high temperatures (20 to 29°C) to germinate (Sanjel et al. 2019). The initial symptoms of orange rust manifest on the underside of the leaf, appearing as small, oval-elongated lesions with a yellow-orange hue, measuring between 1 and 4 mm long. Within 7 days of infection, these spots evolve into blister-like elongated lesions (Fig. 1A). When the disease progresses, these young lesions mature into pustules or uredinia, reaching lengths up to 4 mm that contain single-celled, obovoid or pyriform urediniospores. Only urediniospores have been seen in Florida. The pustules rupture the leaf epidermis (cuticle), thus releasing the spores that create a big mass of orange-golden-colored spores scattered on the lower surface of the leaf (Comstock et al. 2008; Rott et al. 2014). When the spores are mature, they turn orange-cinnamon brown, and pustules can merge, thus forming necrotic leaf tissue (Fig. 1B). Teliospores have only been seen on some sugarcane varieties but so far not in Florida (Bermúdez Guzmán et al. 2016; Comstock et al. 2008; Dixon et al. 2010). Visual identification of teliospores on the underside of the leaf without magnification is usually not possible. Laboratory techniques such as microscopic observations are needed for accurate identification (Chona and Munjal 1950; Virtudazo et al. 2001). Teliospores are two-celled and rarely three-celled, hyaline, and scarce with thin walls, and they have a clavate shape without constriction at the septum. Rust-orange lesions can cover the whole leaf surface, thus causing a decrease in leaf growth because leaf photosynthesis and transpiration are affected. The leaves may turn yellowish as the disease progresses, causing leaf drop and deformation (Zhao et al. 2011). Rust infection causes reduced stalk growth and decreased production and accumulation of sugar in the stalk (Rott et al. 2014).

    FIGURE 1

    FIGURE 1 Orange rust disease symptoms and signs of Puccinia kuehnii on sugarcane leaves. A, Young sugarcane leaf with dispersed and aggregated orange rust lesions (uredinia). B, Severe disease symptoms of orange rust on an older sugarcane leaf. All photos are courtesy of the authors.

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    Host Range

    P. kuehnii is known to infect cultivated and wild Saccharum species, including Saccharum hybrids (commercial sugarcane), S. edule (lowland pitpit), S. officinarum (noble cane), S. robustum (robust sugarcane), and S. spontaneum (wild sugarcane) (Ryan and Egan 1989). Based on early reports from the 1910s, P. kuehnii also infects other wild cane species such as Erianthus arundinaceus, Narenga sp., and Sclerostachya fuscum (Miscanthus fuscus), but this remains to be confirmed using molecular diagnosis (Egan 1964). There are no reports of an alternate host plant where the orange rust fungus can sexually reproduce. The orange rust fungus can complete its whole life cycle on sugarcane, producing urediniospores in an asexual form.

    Geographic Distribution

    The first report of sugarcane orange rust was done in Java by (W. Krüger) E. J. Butler in 1890, and the pathogen was called Uromyces kuehnii (Table 1; Fig. 2) (Butler 1919). In 1893, Cobb identified U. kuehnii in Australia, and in 1898, it was renamed Uredo kuehnii by Wakker and Went. E. J. Butler collected this fungus in 1912 from S. spontaneum in Burma (now Myanmar) and identified teliospores that lead to the reclassification of the fungus as P. kuehnii in 1918 (Egan 1964). During the early 1900s, urediniospores attributed to sugarcane orange rust were found in different areas in Asia–Oceania, including Japan (1909), India (1914), Tahiti (1916), and the Philippines (1922) (Chona and Munjal 1950). Several years later, P. kuehnii was also reported in additional and various locations, although the precise timeframe remains unclear. The disease was observed before 1964 in China, Fiji, Taiwan, Thailand, and Sri Lanka and before 1980 in Guam, Malaysia, New Caledonia, Pakistan, Papua New Guinea, and Samoa. The Solomon Islands and Vietnam are also among the locations affected by orange rust in the 20th century. Orange rust was considered a minor disease for decades until the most important economic losses occurred in Australia. In the late 1990s with the severe epidemic outbreak on major commercial variety Q124, orange rust caused millions of United States dollars (USD) in financial losses (Magarey et al. 2001; Rott et al. 2014). After this outbreak, orange rust began spreading around different parts of the world. It has been hypothesized that P. kuehnii came to the Western Hemisphere from Africa via transoceanic winds (Comstock et al. 2008; Grisham et al. 2013). Starting in 2007 and within 15 years, the disease was reported in at least 23 new locations in Africa and the Western Hemisphere (Table 1).

    TABLE 1 Chronological observations and geographical distribution of sugarcane orange rust caused by Puccinia kuehnii

    FIGURE 2

    FIGURE 2 Worldwide distribution map of sugarcane orange rust disease. The map is showing the location (dots) where sugarcane orange rust (SOR) has been confirmed on Saccharum species. SOR distribution data were compiled from CABI (https://www.cabi.org/isc/datasheet/45818#tosummaryOfInvasiveness) and the sources are listed in Table 1. Eastern Hemisphere locations (before disease emergence in the Western Hemisphere) are shown in blue dots, and Western Hemisphere locations and locations in Africa (after first report in 2007 in Florida) are indicated with red dots.

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    In the United States, orange rust was first reported in Florida in 2007, when urediniospores of P. kuehnii were found on sugarcane cultivars CP80-1743, CPCL99-177, and CPCL01-1055 (Comstock et al. 2008). The first occurrence of orange rust in Louisiana was reported in 2012 on cultivar Ho05-961 (Grisham et al. 2013) and in 2017 in Texas on cultivar HoCP 14-80 (Grisham et al. 2020). P. kuehnii was also reported in Costa Rica in July 2007 on cultivars SP71-5574, CP72-2086, Pindar, Q132, Q138, and SP79-2233. In Nicaragua, disease symptoms were observed on susceptible cultivar CP72-2086 in August 2007 (Chavarría et al. 2009). One month later in Guatemala, urediniospores were found on cultivars CP72-2086, CG96-52, CG 98-0115, and SP 79-2233 (Ovalle et al. 2008). Over the following years, the disease spread to additional locations, and symptoms were observed in Mexico on 17 July 2008 on cultivars Mex 57-1285, Mex 61-230, and Co 301. In 2008, orange rust was found on cultivar CP72-2086 in El Salvador and cultivar SP74-8355 in Panama (Flores et al. 2009). In Cuba, urediniospores were found on cultivars CSG 86-504, CSG 24-92, CSG 204-92, and CC 87-409 in November 2008 (Pérez-Vicente et al. 2010). In December 2009, leaf lesions were observed in Brazil in a nursery plot of pre-commercial cultivar Centauro, followed by cultivars SP89-1115, RB72454, and SP84-2025 (Barbasso et al. 2010). In July 2010, rust disease symptoms were seen in Colombia on cultivars CC01-1884, CC01-1866, and CC01-1305 (Cadavid et al. 2012). One year later, cultivars CR87339, CR83323, BR9806, BR9816, and BT88133 were affected in the Dominican Republic (Briggs et al. 2014). In August 2011, rust pustules were observed on leaves of cultivar SP79-2233 and other sugarcane clones (EC06-351, EC06-340, and EC01-744) from the breeding program at the Sugarcane Research Center of Ecuador (CINCAE), located on the Ecuador Pacific coast (Garcés et al. 2014). During 2012, uredinial lesions were found in Guyana on cultivar DB7869 (Briggs et al. 2018). After several years, in April 2015, urediniospores were found in Misiones Province, Argentina (Funes et al. 2016).

    New locations affected by sugarcane orange rust were also recently reported in Africa. Disease symptoms were observed in July 2009 in the Ivory Coast on cultivars SP71-6180 and Co997. In 2010, cultivars D88172, FR87482, and RB72-454 and breeding clones RCmr 08/319 and RCmr 08/1121 were symptomatic in Cameroon, thus confirming that the disease was also occurring in western and central Africa (Saumtally et al. 2011). More recently, orange rust was found in Reunion and Mauritius, two islands off the coast of South Africa, on cultivar M2705/06 (Hubert et al. 2019; McFarlane et al. 2023; Mungur et al. 2020). The reasons why the sugarcane orange rust pathogen spread worldwide in the early 2000s, while it was limited to the Asia–Oceania region for decades (Fig. 2), remain unknown. In the late 1990s, the orange rust epidemics on cultivar Q124 in Australia were linked to the emergence of a new race of P. kuehnii. Subsequently, it is speculated that this newly identified race of P. kuehnii was disseminated globally (Rutherford 2018).

    Pathogen Isolation

    P. kuehnii is a biotroph fungal pathogen that cannot be grown on culture media in the laboratory. Fungal spores germinate on the sugarcane leaf and have a reproduction cycle of 10 to 14 days. After disruption of the leaf epidermis (Fig. 3A), hundreds of urediniospores are released from the pustules or uredinia (Fig. 3B). The fungal spores can be harvested from sugarcane leaves using a hand-made glass needle or by vacuuming with a mini cyclone spore collector or a laboratory vacuum pump (Goh 1999; Teng and Close 1977).

    FIGURE 3

    FIGURE 3 A, Disruption of the abaxial leaf surface showing orange pustules containing urediniospores. B, Urediniospores of Puccinia kuehnii observed under the microscope (100× magnification). Note the apical thickening of the spores (arrow). All photos are courtesy of the authors.

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    Pathogen Identification Using Morphological Methods

    Sugarcane is the host of more than 100 diseases, and several pathogens can cause yellow or brown spots or stripes on the leaves during the early stages of disease development (Rott et al. 2000). These pathogens include three rust fungi but also other fungi such as Cercospora longipes (causal agent of brown spot), Bipolaris stenospila (causal agent of brown stripe), B. sacchari (causal agent of eye spot), Epicoccum sorghinum (formerly Leptosphaeria sacchari, causal agent of ring spot), and Passalora koepkei (formerly Mycovellosiella koepkei, causal agent of yellow spot). However, when symptoms progress, only rust pathogens will produce pustules (= uredinia) or blister-like symptoms on the lower side of the leaf blade. These pustules break through the leaf epidermis, giving the surface a rough appearance. Mature pustules will erupt and expose masses of urediniospores. Two of the three rust species occur in Florida (P. melanocephala and P. kuehnii), but the third species (Macruropyxis fulva, causal agent of tawny rust) has only been reported in the southern part of Africa (Rutherford 2018). The three sugarcane rusts can be mistaken for one another based solely on symptoms, but some features can be helpful for diagnosing a particular rust disease of sugarcane. Brown rust pustules are more reddish-brown to dark brown and never orange to orange brown like those of orange rust (Fig. 4A and B). Tawny rust pustules are bright orange to orange red. Pustules of orange rust often occur in clusters on the lower side of the leaf, and lesions are more abundant toward the middle third and the lower base of the leaf blade. Pustules of brown and tawny rust are more spread along the leaf blade and tend to be more abundant at the leaf tip than bottom. Under the microscope, urediniospores of the three rust species have overlapping size ranges. However, spores of P. kuehnii are orange to reddish-brown, while those of P. melanocephala are cinnamon-brown to dark brown, and those of M. fulva are bright orange when fresh (Rutherford 2018). Morphological characterization of the orange rust pathogen, often collected from reddish-brown uredinial lesions, is typically conducted using light optical microscopy (Glynn et al. 2010). Urediniospores should be 33 to 53 × 21 to 31 µm with an obovoid to pyriform ellipsoidal shape (Rutherford 2018). Orange-colored urediniospores of P. kuehnii have a thickened pronounced apical cell wall (1 to 2.3 μm and up to 12 μm) with generally few paraphyses (Fig. 3B), which distinguishes them from spores of P. melanocephala, the brown rust pathogen of sugarcane. Urediniospores of P. melanocephala have a brown coloration, do not show an apical thickening, and have abundant paraphyses (Fig. 4C) (Rott et al. 2014; Rutherford 2018).

    FIGURE 4

    FIGURE 4 Brown rust disease symptoms and signs of Puccinia melanocephala on sugarcane leaves. A, Young sugarcane leaf with scattered cinnamon-brown rust lesions (uredinia). B, Severe disease symptoms of brown rust with scattered cinnamon-brown rust lesions (uredinia). C, Urediniospores of P. melanocephala observed under the microscope (100× magnification). All photos are courtesy of the authors.

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    Pathogen Identification Using Molecular Methods

    To date, no reference genome is available for P. kuehnii or the two other rust species infecting sugarcane. Several sequencing projects of the three sugarcane rusts are in progress and should yield valuable genome resources in the coming years. However, the internal transcribed spacer (ITS) region has been partially sequenced for more than 50 isolates of P. kuehnii based on universal primers for this region. Sequences of the ITS region have been used to develop nucleic acid-based diagnostic assays such as polymerase chain reaction (PCR). The ribosomal rDNA-ITS region can be amplified using two primer sets, ensuring accurate diagnosis of P. kuehnii as described by Glynn et al. (2010). Three sets of primers were developed, one to obtain a positive result regardless of the Puccinia species causing sugarcane rust and two others to obtain a positive and a negative result. If a negative result is obtained with all three sets of primers, the rDNA region needs to be amplified and sequenced using universal rust primers as it was performed for tawny rust (Martin et al. 2017). Sugarcane Puccinia rust (P. kuehnii and P. melanocephala)-specific primers PkPmF/PkPmR yield a DNA fragment of 585 and 606 bp for the brown rust and the orange rust pathogens, respectively. The sizes of these fragments cannot be visually differentiated on an agarose electrophoresis gel. Therefore, these DNA fragments must be sequenced and basic local alignment search tool (BLAST) searched against the GenBank database for unequivocal identification. Additionally, P. kuehnii species-specific primers Pk1F/Pk1R yield a PCR amplicon of 527 bp (Table 2) (Glynn et al. 2010). P. kuehnii can also be identified using a real time (RT)-PCR assay with a specific primer pair called Pk2F and Pk2R that generates a 142-bp amplicon. A 136-bp product is obtained when detection is performed with a primer-introduced restriction analysis PCR (PIRA-PCR) assay with primers Pk-PIRAF and Pk-PIRAR (Glynn et al. 2010). Like for conventional PCR, (RT)-amplified DNA products should be sequenced to confirm the identity of P. kuehnii and for the comparison with other P. kuehnii isolates from different geographical locations.

    TABLE 2 List of molecular assays and primers used for identification and detection of Puccinia kuehnii

    Challenges and Limitations of P. kuehnii Diagnosis Methods

    Although morphological methods and molecular methods can be used for the diagnosis of P. kuehnii, they both have their challenges and limitations. Rust pustules can easily be identified in the field on the lower side of sugarcane leaves using the naked eye or with a magnifying glass. However, distinguishing P. kuehnii from the two other rust species infecting sugarcane (P. melanocephala and M. fulva) can be challenging if young and older lesions are not simultaneously present on the diseased plant. Young uredinia and fresh urediniospores have a color that can be associated with each of these rust species. The size and location of the older lesions on the leaf is also indicative of each rust species. Observation of spore morphology and especially looking for the apical thickening of spores of P. kuehnii is another option. However, this requires the use of a microscope, and not all spores exhibit this feature in a sample. Ultimately, only amplification and sequencing of a portion of the rRNA will result in the identification of the pathogen species without a shadow of a doubt. However, nucleic acid-based assays are expensive, time consuming, can only be applied on a limited number of samples, and require an equipped laboratory. They are therefore only used when orange rust is suspected for the first time in a sugarcane growing location in a new variety or when a previously resistant variety shows symptoms in a location already affected by orange rust. Collecting an adequate quantity of spores for DNA extraction can be challenging when only a few plants are diseased and when a limited number of uredinia are available. Nevertheless, it is possible to perform diagnosis of P. kuehnii using only spores from a single uredinium. Clonal rust DNA can be obtained from these spores by the multiple displacement amplification (MDA) method and used for subsequent PCR with specific primers (P. Rott, M. Hincapie, and J. Dijoux, unpublished data; Sanjel et al. 2021).

    Pathogen Storage

    For short-term storage, P. kuehnii urediniospores can be kept at room temperature for 24 to 48 hours in open 0.5- to 2-ml Eppendorf tubes for the spores to dry before inoculation/pathogenicity assays. For long-term storage, dried urediniospores can be stored at room temperature in a desiccator or at −20 or −80°C, although no data are available regarding spore viability after long-term storage. Application of a heat shock for 2.5 min at 42°C to stored spores while leaving the microtube open overnight at room temperature for spore activation and rehydration has been recommended (Braithwaite 2005). This process should be applied before the spore germination test. Long-term stored spores have been successfully used for DNA/RNA extraction and genetic analyses.

    For quantitative molecular investigations, plant leaves infected with P. kuehnii should be processed with liquid nitrogen into a powder and stored at −80°C before nucleic acid extraction. Dried leaf fragments with rust uredinia can also be kept at room temperature in a dry environment, such as a desiccator, for several months and even years before performing PCR assays or other genetic analyses but most likely not for germination or detached leaf assays.

    Pathogenicity Test

    Pathogenicity assays are usually performed using artificial inoculation of entire plants or detached leaf pieces (Moreira et al. 2018; Sanjel et al. 2019; Urashima et al. 2020). Pathogenicity of P. kuehnii can be verified in a greenhouse or in a growth chamber by spraying the sugarcane foliage with a spore suspension, by placing a spore suspension in the leaf whorl of the stalk, or in the laboratory by brushing spores on detached leaf fragments (Chaulagain et al. 2019b; Moreira et al. 2018; Sanjel et al. 2019; Sood et al. 2009). In a greenhouse or in a growth chamber, sprayed plants must be maintained in a high humidity environment for at least 24 h at an average temperature of 22°C to ensure efficient leaf infection by the pathogen (Chapola et al. 2016). Aggressiveness of the pathogen is estimated through disease severity scoring by counting the number of rust lesions or uredinia per square centimeter or per leaf section (Chapola et al. 2016; Sanjel et al. 2019). These methods can also be used to screen sugarcane for resistance to orange rust, and the detached leaf assay also proved to be efficient for testing the efficacy of fungicides to control the disease (Chapola et al. 2016; Chaulagain et al. 2019a; Sood et al. 2009).

    The initial step of any of these inoculation methods consists of collecting the inoculum from symptomatic plants (Fig. 5A). Symptomatic sugarcane leaves are usually taken from the field. Urediniospores of P. kuehnii can be collected from mature pustules on the back side of the leaves (abaxial side) using a glass collector attached to a vacuum pump and can be displaced in Eppendorf tubes (see also the pathogen isolation section above). Spores can also be collected by brushing leaves or shaking the leaves on foil paper. The best collection method to limit microbial contaminants consists of observing the rust lesions under a dissecting microscope and collecting the urediniospores from a single fresh pustule (without other visible microorganisms) with a sterile scalpel, a glass needle, or a pipette tip (Fig. 5B and C). This latter procedure, which provides the cleanest spore samples, is, however, very time-consuming.

    FIGURE 5

    FIGURE 5 Workflow of pathogen isolation and inoculation. A, Collection of infected leaves from the sugarcane field. B, Observation of symptoms (open uredinia or pustules) under the microscope. C, Collection of urediniospores from pustules with a glass collector connected to a vacuum pump or with the tip of a scalpel. D, Preparation of a Puccinia kuehnii suspension adjusted at 105 urediniospores/ml in 0.002% 1-Nonanol supplemented with 0.01% Tween 20. E, Germination assay and visualization of spore germination. F, Inoculation by brushing healthy sugarcane leaves with the suspension of P. kuehnii spores. G, Incubation of inoculated leaf fragments in Magenta vessels under controlled conditions of light and temperature. H, Symptoms of P. kuehnii on the lower surface of sugarcane leaves 10 to 14 days postinoculation. All photos are courtesy of the authors.

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    Once urediniospores are collected, they need to be dried overnight or up to 1 to 2 days at room temperature. It is important to carry out a germination assay to determine the viability of the pathogen as a percentage of spores able to germinate. For that purpose, urediniospores are suspended in a sterile 0.002% 1-Nonanol solution with 0.01% Tween 20 (Fig. 5D). After 10 min, spores should have dropped to the bottom of the solution, and impurities can be discharged from the supernatant. The spore suspension is usually adjusted to 105 urediniospores/ml of the 1-Nonanol/Tween solution using a hemacytometer. The 1-Nonanol is used because it increases spore germination (Sood et al. 2009). The spore germination rate is determined by spreading 10 µl of the spore suspension on a glass slide covered with a 1-mm layer of 1% water agar (Fig. 5E). The covered glass slide should be placed in a sealed container with distilled water on the bottom to enhance humidity to avoid desiccation of the agar. Sporulation and germ tube elongation are visualized under the microscope after a 24-h incubation at 20 to 29°C. A minimum of 100 spores from different fields of the slide should be observed for the presence of a germ tube that is at least the size of the spore. Only rust isolates showing more than 40% viable spore germination should be used for inoculation (Chaulagain et al. 2019a).

    Spore samples with an acceptable spore germination rate can be used for inoculation of sugarcane to test P. kuehnii for pathogenicity (comparison of virulence of different isolates for example) or for screening sugarcane for resistance to orange rust. Inoculum concentration of 105 viable spores/ml must be determined with a hemacytometer and adjusted before inoculation. In all inoculation assays and to optimize infection rates, it is preferable to select young leaves. Young leaves are those located at the top of the sugarcane stalk. The youngest and highest fully emerged leaf can be distinguished by a visible collar at the junction where the leaf blade meets the leaf sheath, and this leaf is called the top visible dewlap (TVD) leaf or the F + 1 leaf. The consecutive leaves below the TVD leaf are known as F + 2, F + 3, and so on (Fig. 6). For the spray inoculation method, young sugarcane leaves are sometimes positioned horizontally to avoid excessive suspension runoff. Up to 6-month-old plants or isolated leaf pieces taken from these plants work best for inoculation assays. The spore inoculum is usually calibrated at 105 viable urediniospores/ml of the Tween/Nonanol solution (Chapola et al. 2016). For the leaf whorl inoculation method, 0.5 ml of the spore Nonanol/Tween suspension is applied with a pipette into the leaf whorl of the stalk (Fig. 7). After inoculation by the leaf-whorl method, it is important to not disturb the plants so that the inoculum applied in the leaf whorl remains in place. Symptoms will start to appear approximately 1 week after inoculation (Sood et al. 2009). For the detached leaf assay, healthy sugarcane leaves are cut into 10-cm pieces and are washed carefully with sterile deionized water (Chaulagain et al. 2019a; Sanjel et al. 2021). The leaf fragments are inoculated by brushing 100 µl of the fungal suspension at 105 viable urediniospores/ml of the Tween/Nonanol solution (Fig. 5F). The inoculated leaf fragments are placed in Magenta vessels (Sigma-Aldrich, Merk Germany) (77 × 77 × 97 mm) with 40 ml of sterile water at the bottom and then in a growth chamber with 8 h of fluorescent light at 25 ± 0.5°C and 16 h of dark at 21 ± 0.5°C (Fig. 5G). Uredinia containing urediniospores will usually develop within 10 to 14 days (Chaulagain et al. 2019a; Sanjel et al. 2021). It is important to note that after 10 days postinoculation, the humidity within the Magenta vessels can create conditions conducive to the growth of opportunistic fungi. Consequently, sugarcane leaves become susceptible to these fungi, leading to the development of an orange hue. Urediniospores can be collected from these lesions to perform another or additional rounds of monospore inoculations to obtain “pure” samples of orange rust spores knowing that rust fungi typically carry other fungi such as ascomycetes and basidiomycetes (Porto et al. 2019; Tao et al. 2019).

    FIGURE 6

    FIGURE 6 Symptoms of sugarcane orange rust caused by Puccinia kuehnii on different leaf positions. TVD leaf or F + 1 leaf = top visible dewlap (TVD) leaf. F + 2 leaf = first leaf below TVD leaf, F + 3 = second leaf below TVD leaf, and so on. A, Cluster of rust uredinia on a TVD leaf; B, older leaf (F + 4 or F + 5) entirely covered with orange rust lesions. All photos are courtesy of the authors.

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    FIGURE 7

    FIGURE 7 Inoculation of sugarcane in the field with Puccinia kuehnii by the leaf whorl method. A, Cut leaf blades (arrows) to identify plants to be inoculated. B, Spore suspension deposited in the leaf whorl of a sugarcane stalk using a syringe. C, Close up of the reaction to orange rust of a susceptible variety 4 weeks postinoculation (4 wpi). D, Reaction to orange rust of a resistant variety 4 wpi. All photos are courtesy of the authors.

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    Regardless of the assay performed, it is essential to inoculate young leaves or leaf sections from the top of the sugarcane plant. Moreover, environmental conditions such as temperature and humidity must be controlled to provide an effective evaluation of pathogenicity when using entire plants. Only temperature (8 h light at 25 ± 0.5°C and 16 h of dark at 21 ± 0.5°C) needs to be controlled when inoculated leaf pieces are placed in Magenta vessels (Sigma-Aldrich, Merk Germany), as those contain water and ensure high humidity for infection. The first symptoms (chlorotic lesions) usually appear 3 to 4 days postinoculation, and uredinia appear 10 to 14 days postinoculation (Fig. 5H). Resistance to orange rust is generally determined in the field after natural infection of entire plants and various scoring systems are used. The percentage of leaf area affected by rust can be determined using the scale (1 to 50%) developed by Purdy and Dean (1981) (Sanjel et al. 2019). Another option includes 0 to 4, 0 to 9, or 1 to 9 scales based on an increasing number of pustules on the leaves (Chapola et al. 2016; Klosowski et al. 2013; Zhao et al. 2015). A whorl inoculation method has also been developed by Sood et al. (2009) to evaluate susceptibility of sugarcane to orange rust. The sugarcane varieties are rated using a 0 to 4 scale, where 0 = no symptoms on the leaf blade, 1 = only chlorotic flecks, 2 = red lesions but no rust sporulation, 3 = many pustules with sporulation (production of urediniospores), and 4 = massive sporulation and necrosis (Fig. 8) (Chapola et al. 2016; Sanjel et al. 2019; Sood et al. 2009). In some other areas, such as Brazil, after spray inoculation, varieties with disease severity scores of 1 to 3 were classified as resistant, varieties with scores from 4 to 6 were classified as intermediate, and varieties with scores from 7 to 9 were classified as susceptible (Fier et al. 2020). When detached leaf pieces of sugarcane are inoculated, rust severity is usually measured by the number of necrotic lesions or the number of uredinia (Sanjel et al. 2021). To ensure the reliability of the experiment, it is necessary to conduct at least three biological replicates, including for negative controls. Negative controls are essential components of the experimental design, typically involving the inoculation of a leaf with either deionized water or a Nonanol solution devoid of urediniospores. These controls are subjected to the same controlled conditions or inoculation process utilized for the samples inoculated with urediniospores, ensuring accurate interpretation of results.

    FIGURE 8

    FIGURE 8 Scoring scale of orange rust after inoculation of sugarcane in the field with Puccinia kuehnii by the leaf whorl method. A, 0 = no symptoms on the leaf blade; B, 1 = only chlorotic flecks; C, 2 = red lesions but no rust sporulation; D, 3 = numerous uredinia (pustules) of orange rust producing urediniospores; and E, 4 = intensive production of sporulating orange rust uredinia associated with leaf necrosis. All photos are courtesy of the authors.

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    Pathogenic Variation

    Sugarcane has a complex polyploid genome, and one sugarcane variety differs from another one by numerous genes. Consequently, there are no isogenic lines for sugarcane, and varieties are vegetatively propagated by stalk pieces (cuttings). Inoculation of four different sugarcane varieties with urediniospores of P. kuehnii collected from two varieties (CL85-1040 and CP89-2143) resulted in the identification of two races of the pathogen in Florida (Sanjel et al. 2021). Variety CL85-1040 was chosen because it was susceptible to orange rust since the first discovery of this disease in Florida in 2007. Variety CP89-2143 was chosen because it was resistant to orange rust until 2011 to 2012 when it showed severe disease symptoms, thus suggesting a resistance breakdown. In the inoculation assays performed by Sanjel et al. (2021), the two isolates of P. kuehnii (1040 from CL85-1040 and 2143 from CP89-2143) produced numerous rust uredinia on healthy leaves of CL85-1040, but P. kuehnii 2143 produced 300 to 500% more rust uredinia than P. kuehnii 1040 on inoculated healthy leaves of CP89-2143 (Sanjel et al. 2021). Based on the work performed by Sanjel et al. (2021), at least two races of P. kuehnii occur in Florida. The orange rust epidemics that developed in the late 1990s in Australia (and mentioned above) have been attributed to the development of a new race of the pathogen but has never been proven experimentally. Consequently, although evidence exists for the occurrence of several races of P. kuehnii, there is still a need for more in-depth investigations of the pathogenic variation of the orange rust causal agent. Screening for resistance to sugarcane orange rust in breeding programs is either performed under natural inoculum pressure or using artificial inoculation methods. In Florida, the spore inoculum to screen new sugarcane varieties is collected from sugarcane varieties such as CP89-2143, which is known to host the most virulent race of P. kuehnii (race 2143). The occurrence of pathogenic variants of P. kuehnii (including races PkSp01-01a4 and PkSp01-01) have also been reported in Brazil (Moreira et al. 2018; Urashima et al. 2020).

    Management

    Managing P. kuehnii disease in sugarcane presents a significant challenge given the diverse array of diseases impacting this crop. Effective disease management strategies encompass both nonchemical and chemical approaches. Nonchemical methods include the utilization of resistant varieties developed through breeding programs, which is a longstanding and successful practice. The genetic basis of resistance to orange rust is not well known, but candidate sugarcane genes and quantitative trait loci (QTLs) associated with disease resistance have recently been reported (Dijoux et al. 2024; Yang et al. 2018, 2019). A genome-wide association study of sugarcane revealed that alleles for resistance to sugarcane orange rust appear to be dominant is S. spontaneum (Dijoux et al. 2024). A molecular marker (G1) linked to an orange rust resistance gene has been reported to have a 73% efficiency in predicting a resistant phenotype (Fier et al. 2020). Consequently, G1 has been suggested to be a valuable tool in sugarcane breeding programs aimed at resistance to orange rust.

    Planting sugarcane varieties with cuttings sourced from clean seed cane nurseries and ensuring the use of healthy planting material are methods often used to control sugarcane diseases, but they are not effective against P. kuehnii. This pathogen does not infect the stalk and is consequently not transmitted through seed cane (stalk cuttings), while healthy material becomes rapidly infected by airborne urediniospores present in the close environment (Rott et al. 2018).

    The management of orange rust in sugarcane, a significant concern in Florida's commercial cultivation, relies heavily on chemical control through fungicide applications, particularly due to the susceptibility of several commercially grown varieties (Table 3) (Raid 2010). Effectively, a combination of two or more anti-fungal active ingredients has proven highly successful in preventing disease onset or halting early fungal growth within plants (Chaulagain et al. 2019a).

    TABLE 3 Rust resistance level of major commercial sugarcane varieties in Florida during 2013 to 2022

    Chemical disease management based on the application of fungicides has been undertaken since the late 2000s by the Florida sugarcane industry to control orange and brown rust epidemics. Sugarcane orange rust epidemics are largely influenced by prevailing weather conditions, and field epidemics are generally considered to be favored by warm and humid conditions. An average nighttime temperature of 20 to 22°C is a good predictor of disease severity in Florida (Chaulagain et al. 2020). Timing is therefore critical for fungicide efficacy, and chemical treatments need to begin at the latest when rust symptoms first appear. The optimal strategy for chemical control of orange rust involves the beginning of fungicide applications during late spring, which is when epidemics of sugarcane orange rust usually start in Florida. Orange rust can be controlled using three to four consecutive fungicide applications separated by 3 to 4 weeks (Chaulagain et al. 2019b). Growers spray varieties known to be susceptible to orange rust every year. So far, no breakdown of fungicide resistance has been reported, most likely because growers try to avoid systematic applications of the same fungicide and use a combination of different molecules.

    Fungicide applications to manage sugarcane rust disease adhere to the restrictions and guidelines set by the Fungicide Resistance Action Committee (FRAC). Registered fungicides include several classes of molecules including strobilurins (pyraclostrobin and azoxystrobin that showed the best efficacy), which belong to group 11 quinone outside inhibitor (QoI) fungicides; triazoles (metconazole and propiconazole) group 3 SBI Class I: demethylation inhibitor (DMI) fungicides; and carboxamides (fluxapyroxad) C2 complex of the succinate-dehydrogenase inhibitor (SDHI) fungicide group (Chaulagain et al. 2019b; FRAC 2024; Raid 2010; Rott et al. 2018). Although these fungicides are effective, their efficacy may fluctuate due to varying environmental conditions such as rainfall and sunlight (Rott et al. 2018). The future releases of sugarcane varieties resistant to orange rust should contribute to the reduction of fungicide applications in commercial production of sugarcane in Florida.

    Acknowledgments

    We thank Leonard Fox and his team at the University of Florida (UF), Institute of Food and Agricultural Sciences (IFAS), Everglades Research and Education Center (EREC) in Belle Glade for the cultivation of the sugarcane varieties used for fungal spore collections. We thank Dr. Matthew VanWeelden and Dr. Hardev Sandhu (UF IFAS EREC) and Wayne Davidson (Florida Sugar Cane League) for the information regarding the resistance level to orange and brown rust of major commercial sugarcane varieties in Florida. We would lastly like to thank Dr. Jose Huguet Tapia for his helpful comments, which highlighted the challenges of working with an obligate fungus and the relevance of working with the P. kuehnii–sugarcane pathosystem.

    The author(s) declare no conflict of interest.

    Literature Cited

    Funding: This work was supported by the Florida Sugar Cane League, the Everglades Research and Education Center (EREC), the Indian River Research and Education Center (IRREC), and the National Institute of Food and Agriculture (NIFA) project FLA-IRC-005637.

    The author(s) declare no conflict of interest.