Evaluating Rhizobacterial Antagonists for Controlling Cercospora beticola and Promoting Growth in Beta vulgaris.

Cercospora beticola Sacc. is an ascomycete pathogen that causes Cercospora leaf spot in sugar beets (Beta vulgaris L.) and other related crops. It can lead to significant yield losses if not effectively managed. This study aimed to assess rhizosphere bacteria from sugar beet soil as a biological control agent against C. beticola and evaluate their effect on B. vulgaris. Following a dual-culture screening, 18 bacteria exhibiting over 50% inhibition were selected, with 6 of them demonstrating more than 80% control. The bacteria were identified by sequencing the 16S rRNA gene, revealing 12 potential species belonging to 6 genera, including Bacillus, which was represented by 4 species. Additionally, the biochemical and molecular properties of the bacteria were characterized in depth, as well as plant growth promotion. PCR analysis of the genes responsible for producing antifungal metabolites revealed that 83%, 78%, 89%, and 56% of the selected bacteria possessed bacillomycin-, iturin-, fengycin-, and surfactin-encoding genes, respectively. Infrared spectroscopy analysis confirmed the presence of a lipopeptide structure in the bacterial supernatant filtrate. Subsequently, the bacteria were assessed for their effect on sugar beet plants in controlled conditions. The bacteria exhibited notable capabilities, promoting growth in both roots and shoots, resulting in significant increases in root length and weight and shoot length. A field experiment with four bacterial candidates demonstrated good performance against C. beticola compared to the difenoconazole fungicide. These bacteria played a significant role in disease control, achieving a maximum efficacy of 77.42%, slightly below the 88.51% efficacy attained with difenoconazole. Additional field trials are necessary to verify the protective and growth-promoting effects of these candidates, whether applied individually, combined in consortia, or integrated with chemical inputs in sugar beet crop production.

Major Pathogens Affecting Carob in the Mediterranean Basin: Current Knowledge and Outlook in Italy.

The main pathogens affecting the carob (Ceratonia siliqua) tree in the Mediterranean basin are described in this overview. The most widespread diseases periodically occurring in carob orchards are powdery mildew (Pseudoidium ceratoniae) and cercospora leaf spot (Pseudocercospora ceratoniae). The causal agents of "black leaf spots" (e.g., Pestalotiopsis, Phyllosticta and Septoria spp.) are responsible for symptoms similar to those previously mentioned for foliar diseases, but are reported in carob orchards at a negligible frequency. Likewise, canker and branch diebacks caused by fungal species belonging to Botryosphaeriaceae are almost never recorded. Among the rots of wood tissues that may compromise old carob specimens, "brown cubical rot" caused by Laetiporus sulphureus is the most widespread and recurrent issue; this pathogen is also well-known for producing edible fruit bodies that are appreciated for pharmaceutical and industrial purposes. On the other hand, "white rots" caused by Fomes and Ganoderma species are less common and reported for the first time in this review. Gall-like protuberances on twigs of uncertain aetiology or tumors on branches associated with Rhizobium radiobacter are described, although these symptoms are seldom detected, as they are also observed for necrotic leaf spots caused by Pseudomonas syringae pv. ciccaronei. A worldwide list of pathogens not yet recorded but at high risk of potential introduction in Italian carob-producing areas is also provided. Finally, concerns related to new phytopathogenic fungi vectored by the invasive Xylosandrus compactus ambrosia beetle are addressed. All the described pathogens could become limiting factors for carob production in the near future, because they could be favored by high-density orchards, the increasing global network of trade exchanges, and the high frequency at which extreme events related to climate change occur globally. Thus, symptoms and signs, causal agents, epidemiology, and, whenever applicable, recommendations for disease prevention and management are provided in this review.

First Report of Cercospora Leaf Spot Caused by Cercospora cf. flagellaris on Lonicera japonica Thunb. in Henan, China.

Honeysuckle flower (Lonicera japonica Thunb.) is a traditional Chinese medicinal plant. It is perennial and widely cultivated in China, Japan and Korea. From late August to October in 2021 and 2022, leaf spots symptoms were observed on L. japonica in different planting fields in Yuzhou, Yuanyang and Fenqiu districts, Henan province, China. The disease incidence was above 85% which reduce photosynthesis. Early disease symptoms appeared as small, circular to elliptical, brown spots on the leaves and later the lesions (1 to 5 mm × 1 to 4 mm) slowly developed yellow haloes. The different brown lesions seldom merge and form larger irregular lesions. Small fragments (3 to 5 mm) of leave tissue were excised from the lesion margins and surface-sterilized in 3% NaClO for 3 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. A total number of 8 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The colonies on PDA were whitish to gray, with cottony aerial mycelium. Conidiophores were fasciculate, olivaceous brown, straight or geniculate, uniform in width, multiseptate, and ranged from 290 to 700 µm (560 µm on average, n = 20). Conidia were hyaline, slightly curved or straight, needle shaped, truncate at the base, and terminal at the tip, 3 to 17-septate, and measuring 150 to 240 µm (180 µm on average, n = 20). The morphological features were consistent with Cercospora cf. flagellaris Ellis & G. Martin (Groenewald et al. 2013). The genomic DNA was extracted using CTAB method. The nuclear ribosomal internal transcribed spacer region (ITS), portions of the actin (ACT), histone H3 (HIS3), and translation elongation factor 1-α (TEF1) genes were amplified using primers ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone and Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006), and EF1-728F/EF1-986R (Carbone and Kohn 1999). The resulting 537-bp ITS, 226-bp ACT, 410-bp HIS3, and 306-bp TEF1 sequences of isolate JDJ002 were deposited in GenBank (accession nos. OR492367, OR548247, OR548248 and OR548248, respectively). Sequence analysis revealed that ITS, ACT, HIS3 and TEF1α sequences exhibited ≥99% of identity with the ITS (KP896013), ACT(KP895965), HIS3(MK991295) and TEF1 (MN180408) sequences of C. cf. flagellaris, respectively. A pathogenicity test was conducted on healthy of L. japonica leaves. The healthy leaves pricked from L. japonica plants, rinsed in autoclaved distilled water three times and dried with distilled filter paper. Then twelve healthy leave were inoculated with a mycelial plug (0.4 cm diameter) harvested from the periphery of two week-old colony. As negative control, leaves inoculated with PDA medium plugs. Inoculated leaves were covered with plastic bags to maintain high relative humidity and incubated at 25°C in growth chamber. After 7 days, the inoculated leaves showed symptoms identical to those observed in the field under natural conditions, whereas negative control remained symptom-free. Re-isolation of the fungus from lesions on inoculated leaves confirmed that the causal agent was C. cf. flagellaris. Pathogenicity tests were repeated three times by the same methods with the same results. To our knowledge, this is the first report of C. cf. flagellaris except Cercospora rhamni Fack., Alternaria alternata, Corynespora cassiicola or Phomopsis sp. causing leave spots on L. japonica in China.

QTL discovery for resistance to black spot and cercospora leaf spot, and defoliation in two interconnected F1 bi-parental tetraploid garden rose populations.

Garden roses are an economically important horticultural crop worldwide, and two major fungal pathogens, black spot (Diplocarpon rosae F.A. Wolf) and cercospora leaf spot of rose (Rosisphaerella rosicola Pass.), affect both the health and ornamental value of the plant. Most studies on black spot disease resistance have focused on diploid germplasm, and little work has been performed on cercospora leaf spot resistance. With the use of newly developed software tools for autopolyploid genetics, two interconnected tetraploid garden rose F1 populations (phenotyped over the course of 3 years) were used for quantitative trait locus (QTL) analysis of black spot and cercospora leaf spot resistance as well as plant defoliation. QTLs for black spot resistance were mapped to linkage groups (LGs) 1-6. QTLs for cercospora resistance and susceptibility were found in LGs 1, 4, and 5 and for defoliation in LGs 1, 3, and 5. The major locus on LG 5 for black spot resistance coincides with the previously discovered Rdr4 locus inherited from Rosa L. 'Radbrite' (Brite Eyes™), the common parent used in these mapping populations. This work is the first report of any QTL for cercospora resistance/susceptibility in tetraploid rose germplasm and the first report of defoliation QTL in roses. A major QTL for cercospora susceptibility coincides with the black spot resistance QTL on LG 5 (Rdr4). A major cercospora resistance QTL was found on LG 1. These populations provide a genetic resource that will further the knowledge base of rose genetics as more traits are studied. Studying more traits from these populations will allow for the stacking of various QTLs for desirable traits.

Genome-Wide Identification of B-Box Gene Family and Expression Analysis Suggest Its Roles in Responses to Cercospora Leaf Spot in Sugar Beet (Beta Vulgaris L.).

The B-box (BBX) protein, which is a zinc-finger protein containing one or two B-box domains, plays a crucial role in the growth and development of plants. Plant B-box genes are generally involved in morphogenesis, the growth of floral organs, and various life activities in response to stress. In this study, the sugar beet B-box genes (hereafter referred to as BvBBXs) were identified by searching the homologous sequences of the Arabidopsis thaliana B-box gene family. The gene structure, protein physicochemical properties, and phylogenetic analysis of these genes were systematically analyzed. In this study, 17 B-box gene family members were identified from the sugar beet genome. A B-box domain can be found in all sugar beet BBX proteins. BvBBXs encode 135 to 517 amino acids with a theoretical isoelectric point of 4.12 to 6.70. Chromosome localization studies revealed that BvBBXs were dispersed across nine sugar beet chromosomes except chromosomes 5 and 7. The sugar beet BBX gene family was divided into five subfamilies using phylogenetic analysis. The gene architectures of subfamily members on the same evolutionary tree branch are quite similar. Light, hormonal, and stress-related cis-acting elements can be found in the promoter region of BvBBXs. The BvBBX gene family was differently expressed in sugar beet following Cercospora leaf spot infection, according to RT-qPCR data. It is shown that the BvBBX gene family may influence how the plant reacts to a pathogen infection.

Insights into Antifungal Mechanisms of Bacillus velezensis S141 against Cercospora Leaf Spot in Mungbean (V. radiata).

Cercospora leaf spot (CLS) is caused by Cercospora canescens and is one of the most important diseases of mungbean (Vigna radiata). Cercospora leaf spot may result in economic loss in production areas. The present study investigated the potential of Bacillus velezensis S141 as a biocontrol agent for C. canescens PAK1 growth on culture plates. Cell-free secretions from a dual culture of S141+PAK1 inhibited fungal growth more than those from a single culture of S141. The biocontrol efficiency of S141 against Cercospora leaf spot on mungbean was then evaluated by spraying. The disease severity of Cercospora leaf spot was significantly reduced in plants treated with S141, with a control efficiency of 83% after 2 days of infection. Comparative transcriptomics and qRT-PCR ana-lyses of S141 during C. canescens inhibition were performed to elucidate the antifungal mechanisms underlying its antifungal activity against Cercospora leaf spot. According to the differentially expressed genes, most up-regulated genes involved in the biosynthetic genes encoding enzymatic hydrolases, including protease, ß-glucanase, and N-acyl glucosaminase, were detected in strain S141 following its interaction. Moreover, genes related to secondary metabolites (surfactin, bacilysin, and bacillomycin D) were up-regulated. Collectively, these results suggest that S141 exhibited strong antifungal activity against C. canescens due to multiple enzymatic hydrolases and secondary metabolites. Therefore, the present study provides insights into the biological network responsible for the antifungal activity of B. velezensis S141 against C. canescens.

Resistance to QoI and DMI Fungicides Does Not Reduce Virulence of C. beticola Isolates in North Central United States.

Cercospora leaf spot (CLS) is a destructive disease limiting sugar beet production and is managed using resistant cultivars, crop rotation, and timely applications of effective fungicides. Since 2016, its causal agent, Cercospora beticola, has been reported to be resistant to quinone outside inhibitors (QoIs) and to have reduced sensitive to demethylation inhibitors (DMIs) in sugar beet growing areas in North Dakota and Minnesota. Isolates of C. beticola resistant to QoIs, DMIs, and both QoIs and DMIs were collected from fields in Foxhome, Minnesota, in 2017. Fitness of these resistant isolates was compared with that of QoI- and DMI-sensitive isolates in laboratory and greenhouse studies. In the lab, mycelial growth, spore production, and spore germination were measured. The results showed that resistant isolates had significantly less mycelial growth and spore production than sensitive isolates, while no significant difference in spore germination was detected. In the greenhouse, six leaf-stage sugar beets were inoculated with a spore suspension made from each resistant group and incubated in separate humidity chambers. CLS disease severity was evaluated visually at 7, 14, and 21 days after inoculation (DAI), and the areas under disease progress curve (AUDPC) were calculated. Resistant isolates had significantly smaller AUDPC but still caused as high disease severity as the sensitive ones at 21 DAI. Although QoI- and/or DMI-resistant isolates had a relatively slower disease development, they still caused high disease severity and need to be factored in disease management practices.

Genome sequencing and annotation of Cercospora sesami, a fungal pathogen causing leaf spot to Sesamum indicum.

Cercospora sesami is a plant pathogen that causes leaf spot disease in sesame plants worldwide. In this study, genome sequence assembly of C. sesami isolate Cers 52-10 (MCC 9069) was generated using native paired-end and mate-pair DNA sequencing based on the Illumina HiSeq 2500 platform. The genome assembly of C. sesami is 34.3 Mb in size with an N50 of 26,222 bp and an average GC content of 53.02%. A total number of 10,872 genes were predicted in this study, out of which 9,712 genes were functionally annotated. Genes assigned to carbohydrate-active enzyme classes were also identified during the study. A total of 80 putative effector candidates were predicted and functionally annotated. The C. sesami genome sequence is available at DDBJ/ENA/GenBank, and other associated information is submitted to Mendeley's data. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03468-4.

Bio-fabrication of Zinc Oxide nanoparticles to rescue Mung Bean against Cercospora leaf spot disease.

Plant disease management using nanotechnology is evolving continuously across the world. The purpose of this study was to determine the effect of different concentrations of green synthesized zinc oxide nanoparticles (ZnO NPs) using Trachyspermum ammi seed extract on Cercospora leaf spot disease in mung bean plants under in-vitro and in-planta conditions. Additionally, the effects on mung bean agronomic and physiological parameters were also assessed. The green synthesized ZnO NPs were characterized using UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and Scanning electron microscopy (SEM). Green synthesized NPs were tested for their ability to inhibit fungal growth at five different concentrations under in-vitro experiment. After 7 days of inoculation, ZnO NPs (1200 ppm) inhibited mycelial growth substantially (89.86% ± 0.70). The in-planta experiment showed statistically significant result of disease control (30% ± 11.54) in response to 1200 ppm ZnO NPs. The same treatment showed statistically significant improvements in shoot length, root length, number of leaves, number of pods, shoot fresh weight (28.62%), shoot dry weight (85.18%), root fresh weight (38.88%), and root dry weight (38.88%) compared to the control. Our findings show that green synthesized ZnO NPs can control Cercospora canescens in mung bean, pointing to their use in plant disease control and growth enhancement.

The Multilateral Efficacy of Chitosan and Trichoderma on Sugar Beet.

The majority of all fungal formulations contain Trichoderma spp., making them effective biological control agents for agriculture. Chitosan, one of the most effective natural biopolymers, was also reported as a plant resistance enhancer and as a biocide against a variety of plant pathogens. An in vitro three-way interaction assay of T. atroviride, chitosan, and important plant pathogens (such as Cercospora beticola and Fusarium oxysporum) revealed a synergistic effect on fungistasis. Furthermore, chitosan coating on Beta vulgaris ssp. vulgaris seeds positively affected the onset and efficiency of germination. We show that priming with T. atroviride spores or chitosan leads to the induced expression of a pathogenesis-related gene (PR-3), but only supplementation of chitosan led to significant upregulation of phytoalexin synthesis (PAL) and oxidative stress-related genes (GST) as a defense response. Repeated foliar application of either agent promoted growth, triggered defense reactions, and reduced incidence of Cercospora leaf spot (CLS) disease in B. vulgaris. Our data suggest that both agents are excellent candidates to replace or assist common fungicides in use. Chitosan triggered the systemic resistance and had a biocidal effect, while T. atroviride mainly induced stress-related defense genes in B. vulgaris. We assume that both agents act synergistically across different signaling pathways, which could be of high relevance for their combinatorial and thus beneficial application on field.

Weather Conditions Conducive for the Early-Season Production and Dispersal of Cercospora beticola Spores in the Great Lakes Region of North America.

In many parts of the world including the Great Lakes region of North America, Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is a major foliar disease of sugar beet (Beta vulgaris). Management of CLS involves an integrated approach which includes the application of fungicides. To guide fungicide application timings, disease prediction models are widely used by sugar beet growers in North America. While these models have generally worked well, they have not included information about pathogen presence. Thus, incorporating spore production and dispersal could make them more effective. The current study used sentinel beets to assess the presence of C. beticola spores in the environment early in the 2017 and 2018 growing seasons. Weather variables including air temperature, relative humidity, rainfall, leaf wetness, wind speed, and solar radiation were collected. These data were used to identify environmental variables that correlated with spore levels during a time when CLS is not generally observed in commercial fields. C. beticola spores were detected during mid-April both years, which is much earlier than previously reported. A correlation was found between spore data and all the weather variables examined during at least one of the two years, except for air temperature. In both years, spore presence was significantly correlated with rainfall (P < 0.0001) as well as relative humidity (P < 0.0090). Rainfall was particularly intriguing, with an adjusted R2 of 0.3135 in 2017 and 0.1652 in 2018. Efforts are ongoing to investigate information on spore presence to improve prediction models and CLS management.

First Report of Cercospora apiicola Causing Leaf Spots and Stem Lesions on Celery in China.

Celery (Apium graveolens L.) is a vegetable crop cultivated widely in the Mediterranean, Europe and parts of Asia. From March to May in 2014, leaf spots and stem lesions were observed on celery plants in Yanqing (116°03'E, 40°32'N), Beijing and Chengdu (104°06'E, 30°67'N), Sichuan Province. Plants developed 0.3-1.8 cm diameter subcircular leaf spots with brown centers surrounded by pale yellow halos. Spots on leaves were amphigenous. Necrotic areas on stems were subcircular to elongated, pale brown to brown. Plants in five greenhouses were surveyed with 30 to 60% disease incidence. Necrotic tissue from 8 stems and 12 leaves were cut from the margins of lesions and divided into two parts. One part was treated with lactophenol and used for microscopic examination. The other part was surface sterilized with 4% sodium hypochlorite for 2 min, rinsed three times in sterile water, placed onto 2% malt extract agar (MEA), and incubated at 26°C for seven days with natural daylight. Stromata on leaves and stems were not well developed. Four-to-ten conidiophores (15.3-56.5 × 2.8-5.5 µm) formed in fascicles, emerged through stomata or erupted through the cuticle. Conidia (n=50) were 60-135 × 2.5-4.5 µm, solitary, septate, cylindrical to obclavate-cylindrical, hila thickened and darkened. Colonies were white to smoke-gray, and aerial mycelia were sparse to moderate. Morphological characteristics of the pathogen were similar to Cercospora apiicola (Groenewald et al. 2006; Groenewald et al. 2013). The gDNA of 20 isolates was extracted from mycelium using the Plant Genomic DNA Kit (Tiangen, China). The internal transcribed spacers (ITS), actin (ACT), translation elongation factor 1-α (TEF1) and histone H3 (HIS3) regions were amplified with primer pairs ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone and Kohn 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006). Phylogenetic analysis of multiple genes (Bakhshi et al. 2018) was conducted with the neighbor-joining method using MEGA7. The sequences of our isolate (QC14030702) and five published sequences of C. apiicola were clustered into one clade with a 99% confidence level. The sequences of QC14030702 have been deposited in GenBank with accessions KU870468 for ITS, KU870469 for ACT, KU870470 for TEF1, and KU870471 for HIS3. Pathogenicity of the isolates was tested on plants (cv. Jia Yuan Xi Yang Qin). Because the pathogen sporulated poorly on various media, mycelial fragments were sprayed on leaves in a suspension of 1×106 mL-1 in a greenhouse (temperature 26±0.5°C; RH 98%; photoperiod 12 h). Healthy plants were sprayed with sterilized water as controls. Three replicates of every isolate were conducted, and each replicate included 5 celery plants. After 7 days, leaf spots appeared on all inoculated plants, which were similar to those on celery in the field. All control plants remained asymptomatic. Re-isolation of the fungus from infected tissues showed same morphological and cultural characteristics of C. apiicola as the original isolates. C. apiicola has been reported in Greece, Korea, South Korea and Venezuela on celery, but never been reported in China (Farr and Rossman 2020). C. apiicola potential threatens celery production, and this the first report of the disease in China.

Rapid Detection of Cercospora beticola in Sugar Beet and Mutations Associated with Fungicide Resistance Using LAMP or Probe-Based qPCR.

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most destructive disease of sugar beet worldwide. Although growing CLS-tolerant varieties is helpful, disease management currently requires timely application of fungicides. However, overreliance on fungicides has led to the emergence of fungicide resistance in many C. beticola populations, resulting in multiple epidemics in recent years. Therefore, this study focused on developing a fungicide resistance detection "toolbox" for early detection of C. beticola in sugar beet leaves and mutations associated with different fungicides in the pathogen population. A loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of C. beticola in infected sugar beet leaves. The LAMP primers specific to C. beticola (Cb-LAMP) assay was able to detect C. beticola in inoculated sugar beet leaves as early as 1 day postinoculation. A quinone outside inhibitor (QoI)-LAMP assay was also developed to detect the G143A mutation in cytochrome b associated with QoI resistance in C. beticola. The assay detected the mutation in C. beticola both in vitro and in planta with 100% accuracy. We also developed a probe-based quantitative PCR (qPCR) assay for detecting an E198A mutation in ß-tubulin associated with benzimidazole resistance and a probe-based qPCR assay for detection of mutations in cytochrome P450-dependent sterol 14α-demethylase (Cyp51) associated with resistance to sterol demethylation inhibitor fungicides. The primers and probes used in the assay were highly efficient and precise in differentiating the corresponding fungicide-resistant mutants from sensitive wild-type isolates.

Sensory assessment of Cercospora beticola sporulation for phenotyping the partial disease resistance of sugar beet genotypes.

BACKGROUND: Due to its high damaging potential, Cercospora leaf spot (CLS) caused by Cercospora beticola is a continuous threat to sugar beet production worldwide. Breeding for disease resistance is hampered by the quantitative nature of resistance which may result from differences in penetration, colonization, and sporulation of the pathogen on sugar beet genotypes. In particular, problems in the quantitative assessment of C. beticola sporulation have resulted in the common practice to assess field resistance late in the growth period as quantitative resistance parameter. Recently, hyperspectral sensors have shown potential to assess differences in CLS severity. Hyperspectral microscopy was used for the quantification of C. beticola sporulation on sugar beet leaves in order to characterize the host plant suitability / resistance of genotypes for decision-making in breeding for CLS resistance. RESULTS: Assays with attached and detached leaves demonstrated that vital plant tissue is essential for the full potential of genotypic mechanisms of disease resistance and susceptibility. Spectral information (400 to 900 nm, 160 wavebands) of CLSs recorded before and after induction of C. beticola sporulation allowed the identification of sporulating leaf spot sub-areas. A supervised classification and quantification of sporulation structures was possible, but the necessity of genotype-specific reference spectra restricts the general applicability of this approach. Fungal sporulation could be quantified independent of the host plant genotype by calculating the area under the difference reflection spectrum from hyperspectral imaging before and with sporulation. The overall relationship between sensor-based and visual quantification of C. beticola sporulation on five genotypes differing in CLS resistance was R2 = 0.81; count-based differences among genotypes could be reproduced spectrally. CONCLUSIONS: For the first time, hyperspectral imaging was successfully tested for the quantification of sporulation as a fungal activity depending on host plant suitability. The potential of this non-invasive and non-destructive approach for the quantification of fungal sporulation in other host-pathogen systems and for the phenotyping of crop traits complex as sporulation resistance is discussed.

NMR-Based Metabolic Profiling of Field-Grown Leaves from Sugar Beet Plants Harbouring Different Levels of Resistance to Cercospora Leaf Spot Disease.

Cercospora leaf spot (CLS) is one of the most serious leaf diseases for sugar beet (Beta vulgaris L.) worldwide. The breeding of sugar beet cultivars with both high CLS resistance and high yield is a major challenge for breeders. In this study, we report the nuclear magnetic resonance (NMR)-based metabolic profiling of field-grown leaves for a subset of sugar beet genotypes harbouring different levels of CLS resistance. Leaves were collected from 12 sugar beet genotypes at four time points: seedling, early growth, root enlargement, and disease development stages. ¹H-NMR spectra of foliar metabolites soluble in a deuterium-oxide (D2O)-based buffer were acquired and subjected to multivariate analyses. A principal component analysis (PCA) of the NMR data from the sugar beet leaves shows clear differences among the growth stages. At the later time points, the sugar and glycine betaine contents were increased, whereas the choline content was decreased. The relationship between the foliar metabolite profiles and resistance level to CLS was examined by combining partial least squares projection to latent structure (PLS) or orthogonal PLS (OPLS) analysis and univariate analyses. It was difficult to build a robust model for predicting precisely the disease severity indices (DSIs) of each genotype; however, GABA and Gln differentiated susceptible genotypes (genotypes with weak resistance) from resistant genotypes (genotypes with resistance greater than a moderate level) before inoculation tests. The results suggested that breeders might exclude susceptible genotypes from breeding programs based on foliar metabolites profiled without inoculation tests, which require an enormous amount of time and effort.

Magnetic resonance imaging of sugar beet taproots in soil reveals growth reduction and morphological changes during foliar Cercospora beticola infestation.

Cercospora leaf spot (CLS) infection can cause severe yield loss in sugar beet. Introduction of Cercospora-resistant varieties in breeding programmes is important for plant protection to reduce both fungicide applications and the risk of the development of fungal resistance. However, in vivo monitoring of the sugar-containing taproots at early stages of foliar symptoms and the characterization of the temporal development of disease progression has proven difficult. Non-invasive magnetic resonance imaging (MRI) measurements were conducted to quantify taproot development of genotypes with high (HS) and low (LS) levels of susceptibility after foliar Cercospora inoculation. Fourteen days post-inoculation (dpi) the ratio of infected leaf area was still low (~7%) in both the HS and LS genotypes. However, during this period, the volumetric growth of the taproot had already started to decrease. Additionally, inoculated plants showed a reduction of the increase in width of inner cambial rings while the width of outer rings increased slightly compared with non-inoculated plants. This response partly compensated for the reduced development of inner rings that had a vascular connection with Cercospora-inoculated leaves. Hence, alterations in taproot anatomical features such as volume and cambial ring development can be non-invasively detected already at 14 dpi, providing information on the early impact of the infection on whole-plant performance. All these findings show that MRI is a suitable tool to identify promising candidate parent lines with improved resistance to Cercospora, for example with comparatively lower taproot growth reduction at early stages of canopy infection, for future introduction into breeing programmes.

Óleo de melaleuca (Melaleuca alternifolia Maiden & Betche, Cheel) no controle de cercosporiose em beterraba / Tea tree oil (Melaleuca alternifolia Maiden& Betche, Cheel) in control of Cercospora beticola Sacc. on Beet

RESUMO O óleo volátil da melaleuca (Melaleuca alternifolia Maiden & Betche, Cheel) possui atividade antimicrobiana podendo causar efeitos sobre as plantas. Avaliou-se a inibição do óleo em Cercospora beticolaSacc., e seu efeito no aumento da produção e qualidade de raízes de beterraba. As doses foram de 0,13; 0,67; 0,80 e 1,00% do óleo, além das testemunhas composta pelo meio de cultura Batata Dextrose Ágar (BDA) no experimento in vitro, e água no experimento in vivo. As plantas foram pulverizadas duas vezes por semana. O delineamento foi inteiramente casualizado, com 4 repetições, e as médias foram comparadas pelo teste Tukey a 5% de probabilidade. O índice de infecção das folhas foi determinado por escala diagramática além do peso e diâmetro das raízes. Os resultados de inibição do crescimento micelial para as doses do óleo foram 0; 56; 87; 83 e 99%, e os índices de infecção: 77,08; 35,62; 21,04; 19,37 e 20,00%, respectivamente, para a testemunha e as doses 0,13; 0,67; 0,80 e 1,00% do óleo. Somente na concentração de 0,80% o óleo proporcionou relação positiva entre o ganho de peso e o diâmetro das raízes. O óleo de Melaleuca foi eficaz no controle de C. beticola e, como consequência, houve produção de raízes de beterraba com melhor desenvolvimento.

ABSTRACT The volatile oil from Melaleuca (Melaleuca alternifolia Maiden & Betche Cheel.) has antimicrobial properties and can promote several effects on plant cultivation. The aim of this study was to evaluate the inhibition of the oil in Cercospora beticola Sacc. and if it favors the growth and development of beet root. The doses were 0.13, 0.67, 0.8 and 1% of oil, besides the control PDA (potato-dextrose-agar) in vitro (laboratory condition) and with water as treatment control in vivo (field conditions). The plants were sprayed twice a week. The treatments were completely randomized and the averages were compared using the Tukey test at 5%. The infection rate of leaves was measured by diagrammatic scale besides the weight and diameter of tubers. The inhibition results of the radial growth by oil treatments were 0; 56, 87, 83 and 99%, while the infection rate showed: 77.08, 35.62, 21.04, 19.37 and 20% respectively to the control and to the oil concentration of 0,13; 0,67; 0,80 e 1,00%. Only at concentration of 0.8% the tea tree oil showed a positive relationship between tuber´s weight and tuber´s diameter gains. It can be concluded that tea tree oil is effective to controlling C. beticola, and also promotes an increase on development in beet tubers.

Identification and Precise Mapping of Resistant QTLs of Cercospora Leaf Spot Resistance in Sugar Beet (Beta vulgaris L.).

The complex inheritance of resistance to Cercospora leaf spot (CLS), the most severe fungal foliar disease in sugar beet, was investigated by means of quantitative trait loci (QTL) analysis. Over a three year period, recombinant inbred lines (RILs) of sugar beet (Beta vulgaris L.), generated through a cross between lines resistant ('NK-310mm-O') and susceptible ('NK-184mm-O') to CLS, were field-tested for their resistance to the pathogen. Composite interval mapping (CIM) showed four QTL involved in CLS resistance to be consistently detected. Two resistant QTL (qcr1 on chromosome III, qcr4 on chromosome IX) bearing 'NK-310mm-O' derived alleles promoted resistance. Across 11 investigations, the qcr1 and qcr4 QTL explained approximately 10% and over 20%, respectively, of the variance in the resistance index. Two further QTL (qcr2 on chromosome IV, qcr3 on chromosome VI) bearing 'NK-184mm-O' derived alleles each explained about 10% of the variance. To identify the monogenic effect of the resistance, two QTL derived from 'NK-310mm-O' against the genetic background of 'NK-184mm-O', using molecular markers. The qcr1 and qcr4 were precisely mapped as single QTL, using progenies BC(5)F(1) and BC(2)F(1), respectively. The qcr1 that was located near e11m36-8 had CLS disease severity indices (DSI) about 15% lower than plants homozygous for the 'NK-184mm-O' genotype. As with qcr1, heterozygosis of the qcr4 that was located near e17m47-81 reduced DSI by about 45% compared to homozygosis. These two resistant QTL might be of particular value in marker-assisted selection (MAS) programs in CLS resistance progression.

Comparing Disease Forecasters for Timing Fungicide Sprays to Control Foliar Blight on Carrot.

Alternaria dauci and Cercospora carotae cause foliar blight on carrot and can reduce yield in severely blighted fields. Historically, fungicides are applied every 7 to 14 days even though applications may be made when environmental conditions do not favor blight development. The purpose of this study was to compare a calendar-based application schedule with three disease forecasting systems for timing fungicide sprays to limit foliar blight, and included (i) an A. dauci disease forecaster, (ii) TOM-CAST, using a threshold of 15 disease severity values, and (iii) a disease forecaster developed to control C. apii on celery. Chlorothalonil was applied weekly or according to the forecasting systems to blight-susceptible 'Cellobunch' carrot plants in 2001 and 2002. Overall petiole health was poor ≥8.3; 10 = 100% petiole necrosis) when fungicides were not used. Although all disease forecasters maintained petiole health (≤5.3; 1 = healthy and vigorous), the TOM-CAST program had the best petiole health rating each year (≤2.8). TOM-CAST prompted 38 to 54% fewer applications than the weekly application schedule, resulting in a fungicide savings of $105 and $147/ha in 2001 and 2002, respectively, while providing similar blight control. The number of sprays also was reduced when fungicides were applied according to the A. dauci and C. apii forecasters, but acceptable blight control was not always achieved.

Control of Benzimidazole- and DMI-Resistant Strains of Cercospora beticola with Strobilurin Fungicides.

The control efficacy of two new strobilurin fungicides, trifloxystrobin and pyraclostrobin, against Cercospora beticola isolates resistant and sensitive to sterol demethylation-inhibiting (DMI) fungicides and benzimidazole fungicides and the effects on evolution of resistance were tested in the current study. Control efficacy of strobilurin fungicides was measured using three C. beticola isolates, one DMI-resistant (DMIR), one benzimidazole-resistant (BENR), and one of wild-type sensitivity (WCB). Both pyraclostrobin and trifloxystrobin provided satisfactory control of all the three isolates used in the study, when applied at 5 µg ml-1 and very high levels of control when applied at 10 µg ml-1. Control was independent of the isolate sensitivity to benomyl and difenoconazole. In contrast, benomyl applied at 10 µg ml-1 failed to control sufficiently the benzimidazole-resistant isolate, whereas difenoconazole applied at either 5 or 10 µg ml-1 failed to provide satisfactory control of the DMI-resistant isolate of the pathogen. The effects of strobilurin fungicide applications on the evolution of resistance to benzimidazole and DMI fungicides were tested under field conditions in a 2-year experiment (2003 to 2004). Applications of either trifloxystrobin or pyraclostrobin provided high levels of disease control during both years of the study, whereas applications of either benomyl or difenoconazole provided a moderate control efficacy. Measurements of resistance frequencies to benomyl and to difenoconazole showed that successive applications of benomyl tended to select for high frequencies of benzimidazole-resistant phenotypes, whereas successive applications of difenoconazole tended to select for high frequencies of DMI-resistant phenotypes. In contrast, applications of either trifloxystrobin or pyraclostrobin prevented an increase of benzimidazole- or DMI-resistant phenotypes compared with the plots treated with benomyl or difenoconazole, respectively, and decreased frequency of resistance compared with untreated control plots.