A Distributed and Wireless Data Acquisition System to Fight Against Sugarcane Orange Rust

Orange rust of sugarcane caused by Puccinia kuehnii was detected in Florida in 2007 (1). It was hypothesized that the pathogen originated from Africa because brown rust of sugarcane (synonym common rust) was introduced to the Western Hemisphere from Africa (3). Requests for rust-infected sugarcane samples were made to several western and central African countries to investigate if orange rust of sugarcane was present but as yet undetected. Orange rust had not previously been reported from western or central Africa. At Zuénoula, Ivory Coast in July 2009, symptoms of sugarcane rust were observed on cvs. SP 71-6180 and Co 997 and appeared distinct to those of brown rust of sugarcane. A year later (May 2010), rust-infected specimens of SP 71-6180 and Co 997 from the same location and also from Borotou in Ivory Coast were sent to the USDA-ARS Systematic Mycology and Microbiology Laboratory in Beltsville, MD for identification. Also in May 2010, sugarcane rust was observed at Mbandjock and Nkoteng in Cameroon on cvs. D 88172, FR 87482, and RB 72-454 and on breeding clones RCmr 08/319 and RCmr 08/1121. All specimens had orange uredinial lesions that ranged from 0.6 to 6.5 mm × 200 to 300 μm and were ellipsoidal to elongate. Urediniospores were consistent with P. kuehnii E.J. Butler observed on specimens from Florida (1). DNA isolated from all samples was successfully amplified with P. kuehnii specific primers targeting ITS1 of rDNA (2). The nuclear large subunit region of rDNA of the rust specimens from Ivory Coast (BPI 881015-881017, GenBank Accession No. HQ666888) and Cameroon (BPI 881010-881014, GenBank Accession Nos. HQ666889-HQ666891) were sequenced. DNA sequences for all were identical to sequences of P. kuehnii and distinct from known sequences of P. melanocephala available in GenBank. To our knowledge, this is the first confirmed report of orange rust of sugarcane in western and central Africa. There is evidence that brown rust of sugarcane was introduced to the Western Hemisphere from this region of Africa (3) making it also the likely source of introduction of orange rust. Further experimentation is required to confirm this hypothesis.

Sugarcane orange rust, caused by the fungus Puccinia kuehnii, is a serious disease of sugarcane. The most effective strategy to combat the disease is to develop resistant cultivars. Phenotypic screening for resistance is laborious, and breeders would benefit from molecular markers linked to the trait. The objective of this research was to identify, via association mapping, markers linked to resistance to orange rust. From the germplasm collection at Canal Point, Florida, 724 genotypes were screened for orange rust resistance via artificial inoculation of field-grown plants. A total of 38 susceptible and 37 resistant genotypes that were at or near the extremes of the population distribution were chosen for genotyping. Genotyping was accomplished via capture sequencing using 32,555 sugarcane probes anchored to the sorghum genome. Several methods were used to account for population structure and/or kinship. The most appropriate methods (determined by the genomic inflation factor) were mixed models and EIGENSTRAT. A total of 38 unique SNPs identified by these methods were then validated on 560 members of the population, using high resolution melting of PCR amplicons. Ten of these markers were statistically significant for the quantitative measure of rust severity, of which nine were also significant for the qualitative measure of ‘status’ (resistant/susceptible). The maximum amount of variation explained by any single marker was approximately 11%. This research has provided markers which can be deployed to help select superior parents for orange rust resistance, and also provided valuable insight for future research towards genetic control of orange rust in sugarcane.

Orange rust of sugarcane (Saccharum spp. hybrids), caused by Puccinia kuehnii, is a relatively new disease in the Western Hemisphere that substantially reduces yields in susceptible sugarcane genotypes. The objective of this study was to determine the physiological mechanisms of orange rust-induced reductions in sugarcane growth and yield by quantifying effects of the disease on leaf SPAD index (an indication of leaf chlorophyll content), net photosynthetic rate, dark respiration, maximum quantum yield of CO2 assimilation, carbon fixation efficiency, and the relationships between these leaf photosynthetic components and rust disease ratings. Plants growing in pots were inoculated with the orange rust pathogen using a leaf whorl inoculation method. A disease rating was assigned using a scale from 0 to 4 with intervals of 0.5. At disease ratings ≥2, the rust-infected leaf portion of inoculated plants showed significant reductions in SPAD index, maximum quantum yield, carbon fixation efficiency, stomatal conductance, leaf transpiration rate, and net photosynthetic rate; but the rusted portion of the infected leaves had increased intercellular CO2 concentration and leaf dark respiration rate. Although leaf SPAD index, photosynthetic rate, stomatal conductance, and transpiration rate at the rust-infected portion decreased linearly with increased rust rating, the effect of orange rust on photosynthetic rate was much greater than that on stomatal conductance and transpiration. Unlike earlier reports on other crops, reduction in leaf photosynthesis by orange rust under low light was greater than that under high light conditions. These results help improve the understanding of orange rust etiology and physiological bases of sugarcane yield loss caused by orange rust.

Orange rust of sugarcane caused by Puccinia kuehnii was first reported in Florida in 2007. Since then, several sugarcane cultivars that were resistant during the initial epidemics became susceptible within a few years. These shifts in resistance were attributed to the evolution of the pathogen and appearance of new races. To study the variation in virulence of P. kuehnii, healthy leaf pieces of sugarcane cultivars susceptible to orange rust were brush inoculated with isolates of P. kuehnii collected from susceptible cultivars in the field. After inoculation, leaf pieces were placed in an incubator and disease severity based on the number of rust uredinia was determined 2 weeks postinoculation. Isolates of P. kuehnii collected from sugarcane cultivar CP 89‐2143, which only showed severe symptoms of orange rust starting in 2011–2012, produced 300%–500% more uredinia on CP 89‐2143 than the isolates collected from cultivar CL 85‐1040 that has been susceptible since 2007. Sugarcane cultivar CL 85‐1040 exhibited high and equivalent numbers of uredinia regardless of the inoculated isolate of the pathogen. These data support the occurrence of pathogenic specialization within P. kuehnii and the existence of at least two races of this pathogen in Florida. Analysis of amplified fragment‐length polymorphism among isolates of P. kuehnii from cultivars CP 89‐2143 and CL 85‐1040 differing in resistance to orange rust revealed genetic variation among rust uredinia. However, this variation was not associated with a specific sugarcane cultivar, suggesting that pathogenic variation was not linked to major, but rather to small genetic changes within the genome of P. kuehnii.

RESUMO Devido à escassez de estudos epidemiológicos da ferrugem alaranjada da cana-de-açúcar no Brasil e a importância dessa cultura no agronegócio brasileiro, objetivou-se neste trabalho determinar a influência da temperatura e do período de molhamento foliar na epidemiologia dessa doença. Foram utilizadas plantas da cultivar SP89-1115 com vinte dias de idade inoculadas com o fungo Puccinia kuehnii na concentração de 2 x104 de uredósporos/mL. O delineamento experimental utilizado foi inteiramente casualizado (DIC) em esquema fatorial 5x7 sendo cinco temperaturas (17,5 ; 20,0 ; 22,5 ; 25,0 e 27,5°C) e sete períodos de molhamento foliar (0 ; 4; 8; 12; 16; 20 e 24 horas), com três repetições. As melhores temperaturas para o desenvolvimento da ferrugem alaranjada foram 20,0; 22,5 e 25,0°C sendo esta última a melhor. Nas temperaturas de 22,5 e 25,0°C com 20 horas de molhamento foliar foram observadas que as pústulas apresentaram-se em maior número decrescendo com 24 horas de molhamento. Não foi observado sintomas da doença nos períodos de molhamentos foliares de 0, 4 e 8 horas em todas as temperaturas testadas. Também não ocorreu a doença na temperatura de 27,5°C em todas as combinações dos períodos de molhamentos foliares testados. Estudos de epidemiologia dessa doença são importantes para traçar estratégias de controle e futuramente desenvolver um sistema de previsão para a ferrugem alaranjada da cana-de-açúcar.

Brazil is the world’s largest producer of sugarcane (Saccharum officinarum), but the occurrence and severity of diseases such as orange rust (Puccinia kuehnii), ring spot (Leptosphaeria sacchari), and red rot (Colletotrichum falcatum) could be part of several factors limiting its production and is the reason for replacing cultivars. It is important to use other forms of disease control depending on the time required to obtain new resistant varieties. The use of silicon (Si) can provide more resistance to the plant making its less vulnerable to diseases. A study was conducted under greenhouse and field conditions. The greenhouse study had 13 treatments (control, fungicide, foliar solution I and II, K silicate, silicic acid at 20, 40 and 60 mg ha-1, wollastonite, agrosilicio, wollastonite + fungicide, wollastonite + K silicate; agrosilicio + fungicide; and agrosilicio + K silicate) with four replications. The field study had nine treatments (control, fungicide, foliar solution I and II, K silicate, silicic acid at 100 and 300 g ha-1 and phosphite at 0.5 and 1 L ha-1) and with three replications. Both studies used a randomized block design. The greenhouse study showed an increase in dry mass of pre-sprouted sugarcane seedling and Si uptake with foliar treatments, especially with silicic acid. K silicate and silicic acid showed lower severity of orange rust than the control and fungicide. On the field study silicic acid at 100 g ha-1 and 300 g ha-1 was more efficient in reducing the area under the disease progress curve (AUDPC) of ring spot compared to the control and K silicate. Fungicide was also more efficient in reducing AUDPC compared to the control as well.

With the epidemic onset of the sugarcane orange rust, fungicide applications of the Strobilurins and Triazoles groups have become necessary in susceptible varieties. This study aimed at evaluating the operational conditions of fungicide application in sugarcane (SP81-3250 variety), in relation to the spray deposition on the upper canopy and the effectiveness of the orange rust control. Treatments consisted of two application volumes (30 L ha-1 or 40 L ha-1) and three nozzle deflection angles (0 º, 90 º or 135 º), plus a ground application at the rate of 200 L ha-1, sprayed with a uniform flat spray nozzle of air induction. The ground application resulted in the smallest and the greatest spray deposition on sugarcane leaves and on the soil, respectively. The aerial application at the rate of 30 L ha-1, sprayed by hollow cone nozzles, at a deflection angle of 135 º, provided the best sugarcane orange rust control.

Puccinia kuehnii is an obligate biotrophic fungal pathogen that causes orange rust of sugarcane, which is prevalent in many countries around the globe. In the United States, orange rust was first detected in sugarcane in Florida in 2007 and poses a persistent and economically damaging threat to the sugarcane industry in this region. Here, we generated the first genome assemblies for two isolates of P. kuehnii (1040 and 2143) collected in Florida in 2017 from two sugarcane cultivars, CL85-1040 and CP89-2143, respectively. These two rust genome resources will be of immense value for future genomic studies, particularly further exploration of the predicted secretomes that may help define key pathogenicity determinants for this economically important pathogen.

Orange rust (OR) and brown rust (BR) are among the most important diseases of sugarcane. The objective of this work was to evaluate the efficiency of fungicides in the control of both rusts. Two experiments, one for each rust, were conducted in the state of Parana, Brazil, in plant cane and in ratoon. A randomized block design with 8 treatments and three replications was used. The treatments were: control (T1), pyraclostrobin (T2), pyraclostrobin + epoxiconazole (T3), azoxystrobin + cyproconazole (T4), azoxystrobin + tebuconazole (T5), trifloxystrobin + tebuconazole (T6), azoxystrobin + benzovindiflupyr (T7) and pyraclostrobin + fluxapyroxad (T8). The cultivars RB72454 and RB835486 were used for their susceptibility to OR and BR, respectively. Four applications of fungicides were carried out in plant cane and also in ratoon, in a 30-day intervals. With the severity data over time, the relative area under the disease progress curve (rAUDPC) was calculated. After harvest, yield (tons of cane per hectare-TCH), yield recovery compared to the control (YR) (%), pol% cane (PC) and tons of pol per hectare (TPH) were obtained. From rAUDPC results, treatments T3, T4, T5, and T7 were the most efficient for the control of OR and treatments T2, T3, T4, T5, T7 and T8 were the most efficient for controlling BR. Yield was affected by treatments only in ratoon in both experiments. The YR ranged from 18.5% to 31.1% and from 16.1% to 37.7% to OR and BR, respectively. There were no differences between treatments for PC in both experiments and TPH was influenced by treatments in ratoon in the BR experiment.

Sugarcane orange rust caused by Puccinia kuehnii has recently become an important disease in sugarcane crops and its spread is causing great concern to growers. In this study, we analyzed spores from symptomatic orange rust sugarcane leaves collected in multiple locations in Cuba in a 4-year-period in order to characterize morphological traits of P. kuehnii, establish an adequate molecular technique to characterize it, and determine its infection court in sugarcane. Orange rust caused by P. kuehnii was confirmed by polymerase chain reaction (PCR) and morphological characterization. AFLP markers detected high diversity in P. kuenhnii samples. Sequencing of rDNA regions, as expected, did not reveal differences and SSR markers designed for P. melanocephala could not be transferred to P. kuehnii. In addition to stomata, entry through prickles was also detected as a new infection court in sugarcane. Although the presence of pustules on the adaxial leaf surface was frequently detected, no clear correlation between this presence and density of stomata and/or prickles was found.

Sugarcane orange rust was recently introduced into Brazil and its control is based on the use of resistant varieties. This study aimed to determine the reaction of Brazilian sugarcane varieties to the disease in the field and to compare artificial inoculation methods. Rust severity was assessed in 17 varieties at a 15-day interval. The maximum disease severity (MS%) and the area under the disease progress curve (AUDPC) were determined for each genotype. The artificial inoculation methods tested were: spraying of a spore suspension on 60-day-old plants in the greenhouse, or placing the spore suspension into the leaf whorl of 5-month-old field-grown plants. Nine out of the 17 varieties studied were resistant to the disease, including the most widely grown in new plantings, RB867515 and RB966928. Varieties RB72454, SP89-1115 and SP79-2233 were susceptible, while RB925211 and SP81-3250 were moderately susceptible. Varieties RB855156, RB92579 and SP83-2847 showed an intermediate reaction. Both inoculation methods correlated well with field results. Spray inoculation discriminates better the responses of the varieties and enables the evaluation of more disease variables. Leaf whorl inoculation allows the use of field-grown plants and generates results in a shorter time.

Puccinia kuehnii is a fungal pathogen that causes orange rust in sugarcane, which is now prevalent in many countries. At the early stage of disease, it is almost indistinguishable from brown rust, which is caused by Puccinia melanocephala. Although several PCR assays are available to detect these diseases, the loop-mediated isothermal amplification (LAMP)-based assay has been reported to be more economical and easier to perform. Under isothermal conditions, DNA is amplified with high specificity and rapidity. Moreover, visual judgment of color change without further post-amplification processing makes the method convenient. The present study was undertaken to detect P. kuehnii genomic DNA using four primers corresponding to a unique DNA sequence of P. kuehnii. The LAMP assay was found to be optimal when 8 mM MgSO4 was used and the reaction was incubated at 63 °C for 90 min. Positive samples showed a color change from orange to green upon SYBR Green I dye addition. Specificity of the LAMP test was checked with DNA of P. melanocephala, which showed no reaction. Sensitivity of the LAMP method was observed to be the same as real-time PCR at 0.1 ng, thus providing a rapid and more affordable option for early disease detection.

Orange rust caused by the fungus Puccinia kuehnii greatly affects sugarcane and causes millions of tons of losses in production. This condition was first reported in Brazil at the end of 2009. The disease is currently present in most of the countries that produce this crop. The aim of this research was to develop risk maps of P. kuehnii infection using temperature and relative humidity data, provided by 389 automatic weather stations throughout the country. A spatial distribution analysis was carried out to assess the number of daily hours of favorable conditions for spore germination in each region. In the central-south region, where the main sugarcane producing states are concentrated, two distinct periods were observed during the three years studied. Higher favorability occurred from October to April, and lower favorability occurred from May to September. The opposite relation was observed on the coast of the north-eastern region, where conditions were more favorable to the disease from May to September. The validation data were confirmed by the results of Pearson’s correlation between sugarcane orange rust infection under field conditions and the proposed maps. In conclusion, risk maps obtained using data from automatic weather stations could contribute to the monitoring of the risk of infection by sugarcane orange rust.

Agro-climatic favorability zones for sugarcane orange rust as a tool for cultivar choice and disease management

Symptoms of sugarcane orange rust were first observed in July 2010 on sugarcane (interspecific hybrid of Saccharum L. species) cv. CC 01-1884 planted in the La Cabaña Sugar Mill, Puerto Tejada, Colombia. Morphological features of uredinial lesions and urediniospores inspected with an optical microscope and scanning electron microscopy were distinct from common rust of sugarcane caused by Puccinia melanocephala Syd. & P. Syd., revealing spores identical morphologically to those described for the fungus P. kuehnii (Kruger) E. Butler, causal agent of sugarcane orange rust (1,3). Uredinial lesions were orange and distinctly lighter in color than pustules of P. melanocephala. Urediniospores were orange to light cinnamon brown, mostly ovoid to pyriform, variable in size (27.3 to 39.2 × 16.7 to 21.2 μm), with pronounced apical wall and moderately echinulate with spines evenly distributed. Paraphyses, telia, and teliospores were not observed. Species-specific PCR primers designed from the internal transcribed spacer (ITS)1, ITS2, and 5.8S rDNA regions of P. melanocephala and P. kuehnii were used to differentiate the two species (2). The primers Pm1-F and Pm1-R amplified a 480-bp product from P. melanocepahala DNA in leaf samples with symptoms of common rust. By contrast, the primers Pk1-F and Pk1-R generated a 527-bp product from presumed P. kuehnii DNA in leaf samples with signs of orange rust, confirming the identity as P. kuehnii. The Centro de Investigación de la Caña de Azúcar de Colombia (Cenicaña) started a survey of different cultivars in nurseries and experimental and commercial fields in the Cauca River Valley and collected leaf samples for additional analyses. Experimental cvs. CC 01-1884, CC 01-1866, and CC 01-1305 were found to be highly susceptible to orange rust and were eliminated from regional trials, whereas commercial cvs. CC 85-92 and CC 84-75, the most widely grown cultivars, were resistant. With the discovery of orange rust of sugarcane in Colombia, Cenicaña has incorporated orange rust resistance in the selection and development of new cultivars. To our knowledge, this is the first report of P. kuehnii on sugarcane in Colombia. Orange rust has also been reported from the United States, Cuba, Mexico, Guatemala, Nicaragua, El Salvador, Costa Rica, Panama, Ecuador, and Brazil.