Suppression of Rust and Brown Eye Spot Diseases on Coffee by Phosphites and By‐products of Coffee and Citrus Industries

The rust and brown eye spot, caused by Hemileia vastatrix and Cercospora coffeicola, respectively, are the most important fungal diseases on coffee in South America. Their management is mainly by chemical treatment, and there is no genetic resistance to brown eye spot known so far. Considering the need for developing alternative products for their control, the goal of this work was to evaluate the effects of phosphites and by‐products of coffee and citrus industries on rust and brown eye spot. Formulations of coffee and citrus industry by‐products, phosphites and their combination with fungicide were evaluated in field experiments, and their effect on fungal urediniospores and conidia was evaluated in vitro. In the field, treatments were applied individually or in combination and the in vitro assays were performed with manganese phosphite (Reforce Mn), potassium phosphite and citrus industry by‐product (Fortaleza), copper phosphite and coffee industry by‐product (Fitoforce Full), and fungicide. The severity and incidence of rust and brown eye spot on coffee leaves, yield, and leaf retention were evaluated in the field. Percentage of spore germination was evaluated in vitro for both fungi, whereas mycelial growth was evaluated for C. coffeicola only. The treatments Fortaleza, Reforce Mn and Fitoforce Full suppressed both diseases with a reduction in defoliation. In the year 2012, the plants treated with Reforce Mn and Reforce Mn + Fortaleza showed a yield increase of 72 and 88%, respectively, which was similar to the results shown by the fungicide treatment. In vitro inhibition of germination of H. vastatrix urediniospores and of C. coffeicola conidia was observed and suggests that the products exert some toxic effects to both fungi. Finally, the results observed indicate that the combined use of by‐products of plant‐processing industries and phosphites is an alternative and can be added efficiently to the management of coffee diseases.     

Diagrammatic Scale for Assessment of Bacterial Blight in Coffee Leaves

Due to the lack of a standardized visual method for assessing bacterial blight (Pseudomonas syringae pv. garcae) in coffee leaves, a diagrammatic scale was developed and validated to quantify the disease. Leaves were collected in crops and nursery with different intensity of symptoms, and the true severity was determined electronically. Based on the frequency distribution of severity values and according to the Weber–Fechner's law of visual stimulus, the minimum and maximum limits and the intermediate levels in the scale were determined. Validation was performed by ten evaluators who estimated the severity of 50 leaves with different intensity of symptoms. One evaluation was performed without diagrammatic scale and two evaluations with the scale at 7‐day intervals. The accuracy, precision, repeatability and reproducibility of the estimates were evaluated. The scale had nine levels: 0 (0%), 1 (0.1–0.99%), 2 (1–2%), 3 (2.01–4%), 4 (4.01–8%), 5 (8.01–16%), 6 (16.01–25%), 7 (25.01–45%) and 8 (≥45.1%). Using the scale, the evaluators were able to improve accuracy, precision, reproducibility and repeatability of estimates, compared to evaluators without scale. The scale was appropriate to visual estimation of severity of bacterial blight in coffee leaves.     

Relationship between Incidence of Brown Eye Spot of Coffee Cherries and the Chemical Composition of Coffee Beans

To study the chemical composition of coffee beans from coffee cherries infected by brown eye spot, two experiments were conducted with coffee cherries from Catuaí Amarelo and Acaiá Cerrado farms, in the full physiological maturity stage. The coffee cherries were harvested manually, and 20 litres of cherries without visible symptoms of brown eye spot (healthy coffee cherries) and 20 l of cherries with visible symptoms of the disease (diseased coffee cherries) were individually separated. After separation, the cherries were mixed in five different proportions to form the treatments: 0, 25, 50, 75 and 100% of diseased coffee cherries to 100, 75, 50, 25 and 0% of healthy coffee cherries. The experimental design was performed in randomized blocks, with each 8 l of coffee cherries being considered an experimental unit. After drying (humidity 12%), the chemical characteristics were analysed. Polyphenols, potassium leachate and electrical conductivity had a linear increase with the rising of the proportion of diseased coffee cherries. Total sugars, soluble solids and pH decreased linearly with the rising of the proportion of diseased coffee cherries.     

Silicon and Triadimenol for the Management of Coffee Leaf Rust

This study documents an experiment that was undertaken in the 2006/2007, 2007/2008 and 2008/2009 growing seasons on Coffea arabica cv. ‘Catuaí Vermelho IAC 144’ that sought to evaluate the effects of various calcium silicate rates combined with the fungicide triadimenol on the incidence of coffee leaf rust. The experimental design was a randomized complete block in a split plot with five treatments (with varied calcium silicate rates and with or without triadimenol) and four replications. Each experimental unit (split plot) consisted of seven coffee plants (14 m²), which were the central five plants used for the evaluations. Calcium silicate (CS) and lime (L) were used according to the following mixtures (M): M1: 0% CS and 100% L; M2: 25% CS and 75% L; M3: 50% CS and 50% L; M4: 75% CS and 25% L; and M5: 100% CS and 0% L. The leaf Si concentration did not increase as CS rates increased in the soil. There was no reduction in the area under rust progress curve (AURPC) as the rates of CS increased in the soil. During the growing seasons 2006/2007, 2007/2008 and 2008/2009, rust incidence reached 94, 96 and 92% on plants that did not receive triadimenol, respectively, whereas the incidence did not exceed 6, 38 and 16%, respectively, for those plants that did. For yield, no interaction was observed between the calcium silicate rates and with or without triadimenol. The yield increased by 117% for plants receiving triadimenol compared with those that did not. The 3‐year experiments indicated that soil amendment with calcium silicate had no effect on either reducing coffee leaf rust incidence or increasing yield. Conversely, as expected, coffee leaf rust symptoms were dramatically reduced on plants sprayed with triadimenol, and this was accompanied by a significant gain in yield.     

Effect of Zinc on the Development of Brown Spot in Rice

Brown spot, caused by the fungus Bipolaris oryzae, is one of the most destructive diseases of rice. This study investigated the effect of zinc rates on the development of brown spot in rice. Rice plants (cv. ‘Metica‐1′) were grown in hydroponic culture amended with Zn rates (applied as ZnSO₄^.7H₂O) of 0, 0.5, 1, 2 and 4 μm and inoculated with B. oryzae. The foliar concentration of Zn was determined. Leaf samples were assessed for disease severity, and then, area under brown spot progress curve (AUBSPC) was calculated. The relationship between Zn concentrations on leaf tissues and the rates of this micronutrient was best described by a positive linear regression model, while the relationship between the Zn rates and the AUBSPC was best described with a positive quadratic regression model. The correlation between Zn concentrations on leaf tissues and AUBSPC was positive and significant (r = 0.68, P < 0.05). The results from this study showed that high foliar concentration of Zn was associated with increasing rice susceptibility to brown spot.     

Leaf Spot on Sweet Potato (Ipomoea batatas) Caused by Stemphylium solani,a New Disease in China

During 2010–2011, a severe leaf spot disease of sweet potato (Ipomoea batatas) was found in Haikou City, Hainan province of China. The disease is characterized with large, irregular, brown, necrotic lesions on the margin or in the centre of leaves. A species of Stemphylium was consistently recovered from pieces of symptomatic tissues on PDA. Based on morphological characteristics and molecular identification by rDNA‐ITS gene analysis, the fungal species was identified as Stemphylium solani Weber, and its pathogenicity was confirmed by Koch's postulates. This is the first report of leaf spot on sweet potato caused by S. solani in China.     

Aggressiveness and Genetic Diversity of Hemileia vastatrix During an Epidemic in Colombia

Since 2008, Colombia has been experiencing an epidemic of the coffee rust Hemileia vastatrix. The altitude range of the disease has expanded, and nursery and young plants that were usually not attacked by the disease are now significantly affected. To determine whether this new epidemic has been caused by a new pathogenic isolate, the molecular diversity of the pathogen causing the epidemic in different regions of the country was assessed, using AFLP molecular markers on isolates collected from coffee fields prior and after the year 2008. We also evaluated the aggressiveness of isolates collected from diverse coffee‐producing areas and from different coffee genotypes. Isolates collected before and during the present epidemic were quite similar both genetically and with regard to their aggressiveness. Out of a total of 349 fragments amplified from 6 AFLP primer combinations, 48 (13.2%) were polymorphic and only 18 were unique among H. vastatrix isolates representative of pre‐2008 and post‐2008 epidemic populations. We conclude that the epidemic was caused by the excessive rainfall that has occurred in Colombia since 2006 and that extended to 2011 and not by the arrival of a new isolate of the pathogen or a change in virulence of the species present in the country.     

Severity Evaluation Methods in Common Bean Recurrent Selection Programme for Resistance to Angular Leaf Spot

The objectives of this study were to identify which method and period of evaluation of angular leaf spot (ALS) of common bean, caused by the fungal pathogen Pseudocercospora griseola, allow better discrimination among common bean lines derived from seven cycles of recurrent selection for resistance to this pathogen. For that reason, 35 lines of the first seven cycles of the programme were assessed for disease severity on leaves and pods using a rating scale. For leaves, the methods used were severity in field plots (SF), severity in sampled leaflets (SS) and percentage of the sampled leaf area with symptoms (%LAS). Leaf assessments were performed at 7, 14, 21, 28, 33 and 41 days after flowering (DAF), and area under the disease progress curve (AUDPC) was calculated. On pods, severity was evaluated at 41 DAF. It was observed that the SF using a rating scale is the most efficient method for selection of resistant lines, and the best time period for evaluating the disease is around 33 DAF.     

Red‐Light‐Induced Resistance to Brown Spot Disease Caused by Bipolaris oryzae in Rice

In this study, the protective effect of red light against the brown spot disease caused by the fungus Bipolaris oryzae in rice was investigated. Lesion formation was significantly inhibited on detached leaves that were inoculated with B. oryzae and kept under red for 48 h, but it was not inhibited when the leaves were kept under natural light or in the dark. The protective effect was also observed in intact rice plants inoculated with B. oryzae; the plants survived under red light, but most of them were killed by infection under natural light or dark condition. Red light did not affect fungal infection in onion epidermis cells or heat‐shocked leaves of rice, and it did not affect cellulose digestion ability; this suggested that the protective effect is due to red‐light‐induced resistance. In addition, the degree of protection increased as the red light dosage increased, regardless of the order of the red light and natural light period, indicating that red‐light‐induced resistance is time dependent. Feeding of detached leaves with a tryptophan decarboxylase inhibitor, s‐α‐fluoromethyltryptophan (0.1 mm ), for 24 h inhibited the development of resistance in response to red light irradiation. Suppression of resistance was also observed in leaves treated with a phenylalanine ammonia‐lyase inhibitor, α‐aminooxy acetic acid (0.5 mm ). These results suggest that the tryptophan and phenylpropanoid pathways are involved in the red‐light‐induced resistance of rice to B. oryzae.     

Development and evaluation of a genome‐wide Coffee 8.5K SNP array and its application for high‐density genetic mapping and for investigating the origin of Coffea arabica L.

Coffee species such as Coffea canephora P. (Robusta) and C. arabica L. (Arabica) are important cash crops in tropical regions around the world. C. arabica is an allotetraploid (2n = 4x = 44) originating from a hybridization event of the two diploid species C. canephora and C. eugenioides (2n = 2x = 22). Interestingly, these progenitor species harbour a greater level of genetic variability and are an important source of genes to broaden the narrow Arabica genetic base. Here, we describe the development, evaluation and use of a single‐nucleotide polymorphism (SNP) array for coffee trees. A total of 8580 unique and informative SNPs were selected from C. canephora and C. arabica sequencing data, with 40% of the SNP located in annotated genes. In particular, this array contains 227 markers associated to 149 genes and traits of agronomic importance. Among these, 7065 SNPs (~82.3%) were scorable and evenly distributed over the genome with a mean distance of 54.4 Kb between markers. With this array, we improved the Robusta high‐density genetic map by adding 1307 SNP markers, whereas 945 SNPs were found segregating in the Arabica mapping progeny. A panel of C. canephora accessions was successfully discriminated and over 70% of the SNP markers were transferable across the three species. Furthermore, the canephora‐derived subgenome of C. arabica was shown to be more closely related to C. canephora accessions from northern Uganda than to other current populations. These validated SNP markers and high‐density genetic maps will be useful to molecular genetics and for innovative approaches in coffee breeding.     

Isolation and Identification of Pathogens Causing Marigold (Tagetes erecta L.) Black Spot in Beijing,China

Black spot leads to great marigold losses worldwide. The disease is characterized by black spots on leaves and stems in its early stages, and the whole plant has black rot at the advanced stage. In this report, 6 of 217 Alternaria strains isolated from lesions of marigold plants in Beijing were randomly selected. The morphological characteristics and a pathogenic tree based on two protein‐coding genes (gpd and alt a 1) indicated that Alternaria tagetica is the causal agent of marigold black spot in Beijing. All six Alternaria strains could successfully re‐infect marigold, but they could not infect carrot or zinnia by either spore spray in a greenhouse or planting experiments in the epidemic area. This is the first report of the A. tagetica pathogen being isolated from marigold in Beijing.     

Validation of a cheap and simple nondestructive method for obtaining AFLPs and DNA sequences (mitochondrial and nuclear) in amphibians

The use of nondestructive methods for obtaining DNA from amphibians (e.g. buccal swabs) allows genetic studies to be performed without affecting the survival of the studied individuals. In this study, we compared two methods of nondestructive DNA sampling, buccal swabs and interdigital membrane or toe‐clipping, in several amphibian species of different size: Rhinella spinulosa, R. atacamensis, six species of the genus Telmatobius and Pleurodema thaul. We evaluated the integrity of the DNA extracted by sequencing fragments of mitochondrial and nuclear genes and by generating amplified fragment length polymorphisms markers (AFLPs). In all cases, we obtained an adequate amount of DNA (mean range 55–298 ng/μL). We obtained identical DNA sequences from buccal swab and interdigital membrane/toe‐clip for all individuals. The differences in the coding of AFLP markers between the tissues were similar to those reported for replicas of the same type of sample in similar analyses in other species of amphibians. In conclusion, the use of buccal swabs is a trustworthy and inexpensive method to obtain DNA for mitochondrial and nuclear sequencing and AFLP analyses. Given the types of markers evaluated, buccal swabs may be used for phylogenetic, phylogeographic and population genetic studies, even in small amphibians (<33 mm).     

Bulb and Root Rot in Lily (Lilium longiflorum) and Onion (Allium cepa) in Israel

In the past 10 years, there has been a substantial increase in reports, from growers and extension personnel, on bulb and root rots in lily (Lilium longiflorum) in Israel. Rot in these plants, when grown as cut flowers, caused serious economic damage expressed in reduction in yield and quality. In lily, the fungal pathogens involved in the rot were characterized as binucleate Rhizoctonia AG‐A, Rhizoctonia solani, Pythium oligandrum, Fusarium proliferatum (white and purple isolates) and F. oxysporum, using morphological and molecular criteria. These fungi were the prevalent pathogens in diseased plants collected from commercial greenhouses. Pathogenicity trials were conducted on lily bulbs and onion seedlings under controlled conditions in a greenhouse to complete Koch's postulates. Disease symptoms on lily were most severe in treatments inoculated with binucleate Rhizoctonia AG‐A, P. oligandrum and F. proliferatum. Plant height was lower in the above treatments compared with the control plants. The least aggressive fungus was R. solani. In artificial inoculations of onion, seedling survival was significantly affected by all fungi. The most pathogenic fungus was F. proliferatum w and the least were isolates of F. oxysporum (II and III). All fungi were successfully re‐isolated from the inoculated plants.     

Local and Regional Variation in Local Frequency of Multiple Coffee Pests Across a Mosaic Landscape in Coffea arabica's Native Range

Shaded coffee has been highlighted for its potential to conserve biodiversity, and thus perhaps also a diversity of natural enemies that could control pest organisms. In southwestern Ethiopia, coffee is grown in shade both in contiguous forests and in forest patches with native trees surrounded by open fields. We hypothesized that coffee grown in contiguous forests, which is the natural habitat for coffee (Coffea arabica) and its interacting organisms, would have less pest damage due to high protection by natural enemies. We surveyed pests on coffee plants in plots within contiguous forests (10 sites) and in forest patches (21 sites). In general, the variation in number of damaged or attacked leaves by individual insect or fungal pests was larger between plants than between plots, which suggests that very local conditions or processes are important. The spatial signals were generally weak. Coffee rust and coffee blotch miner tended to have lower infestation rates in accordance with our hypothesis, while fruit flies in ripe berries were more abundant in forest patches closer to contiguous forest. Based on interviews, olive baboons showed a clear dependency on contiguous forest habitat and were regarded as a problem only in contiguous forests and forest patches close to contiguous forests. In conclusion, we found no support for a generally stronger top‐down control on coffee pests in sites within, or with connectivity to, contiguous moist afromontane forests in the native range of coffee.     

Molecular insight into cotton leaf curl geminivirus disease resistance in cultivated cotton (Gossypium hirsutum)

Cultivated cotton (Gossypium hirsutum) is the most important fibre crop in the world. Cotton leaf curl disease (CLCuD) is the major limiting factor and a threat to textile industry in India and Pakistan. All the local cotton cultivars exhibit moderate to no resistance against CLCuD. In this study, we evaluated an exotic cotton accession Mac7 as a resistance source to CLCuD by challenging it with viruliferous whiteflies and performing qPCR to evaluate the presence/absence and relative titre of CLCuD‐associated geminiviruses/betasatellites. The results indicated that replication of pathogenicity determinant betasatellite is significantly attenuated in Mac7 and probably responsible for resistance phenotype. Afterwards, to decipher the genetic basis of CLCuD resistance in Mac7, we performed RNA sequencing on CLCuD‐infested Mac7 and validated RNA‐Seq data with qPCR on 24 independent genes. We performed co‐expression network and pathway analysis for regulation of geminivirus/betasatellite‐interacting genes. We identified nine novel modules with 52 hubs of highly connected genes in network topology within the co‐expression network. Analysis of these hubs indicated the differential regulation of auxin stimulus and cellular localization pathways in response to CLCuD. We also analysed the differential regulation of geminivirus/betasatellite‐interacting genes in Mac7. We further performed the functional validation of selected candidate genes via virus‐induced gene silencing (VIGS). Finally, we evaluated the genomic context of resistance responsive genes and found that these genes are not specific to A or D sub‐genomes of G. hirsutum. These results have important implications in understanding CLCuD resistance mechanism and developing a durable resistance in cultivated cotton.     

Detection and Identification of Aster Yellows Phytoplasma Associated with Lipstick Yellow Frond Disease in Malaysia

Yellowing symptoms similar to coconut yellow decline phytoplasma disease were observed on lipstick palms (Cyrtostachys renda) in Selangor state, Malaysia. Typical symptoms were yellowing, light green fronds, gradual collapse of older fronds and decline in growth. Polymerase chain reaction assay was employed to detect phytoplasma in symptomatic lipstick palms. Extracted DNA was amplified from symptomatic lipstick palms by PCR using phytoplasma‐universal primer pair P1/P7 followed by R16F2n/R16R2. Phytoplasma presence was confirmed, and the 1250 bp products were cloned and sequenced. Sequence analysis indicated that the phytoplasmas associated with lipstick yellow frond disease were isolates of ‘Candidatus Phytoplasma asteris’ belonging to the 16SrI group. Virtual RFLP analysis of the resulting profiles revealed that these palm‐infecting phytoplasmas belong to subgroup 16SrI‐B and a possibly new 16SrI‐subgroup. This is the first report of lipstick palm as a new host of aster yellows phytoplasma (16SrI) in Malaysia and worldwide.     

Comparing concentration‐based (AOT40) and stomatal uptake (PODY) metrics for ozone risk assessment to European forests

Tropospheric ozone (O₃) produces harmful effects to forests and crops, leading to a reduction of land carbon assimilation that, consequently, influences the land sink and the crop yield production. To assess the potential negative O₃ impacts to vegetation, the European Union uses the Accumulated Ozone over Threshold of 40 ppb (AOT40). This index has been chosen for its simplicity and flexibility in handling different ecosystems as well as for its linear relationships with yield or biomass loss. However, AOT40 does not give any information on the physiological O₃ uptake into the leaves since it does not include any environmental constraints to O₃ uptake through stomata. Therefore, an index based on stomatal O₃ uptake (i.e. PODY), which describes the amount of O₃ entering into the leaves, would be more appropriate. Specifically, the PODY metric considers the effects of multiple climatic factors, vegetation characteristics and local and phenological inputs rather than the only atmospheric O₃ concentration. For this reason, the use of PODY in the O₃ risk assessment for vegetation is becoming recommended. We compare different potential O₃ risk assessments based on two methodologies (i.e. AOT40 and stomatal O₃ uptake) using a framework of mesoscale models that produces hourly meteorological and O₃ data at high spatial resolution (12 km) over Europe for the time period 2000–2005. Results indicate a remarkable spatial and temporal inconsistency between the two indices, suggesting that a new definition of European legislative standard is needed in the near future. Besides, our risk assessment based on AOT40 shows a good consistency compared to both in‐situ data and other model‐based datasets. Conversely, risk assessment based on stomatal O₃ uptake shows different spatial patterns compared to other model‐based datasets. This strong inconsistency can be likely related to a different vegetation cover and its associated parameterizations.