Showing papers in "Plant Disease in 2020"

Revisiting Sustainability of Fungicide Seed Treatments for Field Crops

Abstract: The use of fungicide seed treatment (FST) is a very common practice worldwide. The purported effectiveness of many fungicides in providing broad-spectrum and systemic control of important diseases and the perception that FST reduces overall pesticide use, hence lowering environmental impacts, have greatly promoted the use of FST in the last five decades. Since there have been rapid advancements in the types, formulations, and application methods for seed treatments, there is a need to re-evaluate the benefits versus the risks of FST as a practice. While the use of seeds treated with neonicotinoid insecticides has come under scrutiny due to concern over potential nontarget effects, there are knowledge gaps on potential negative impacts of FST on operators' (those who apply, handle, and use treated seeds) health and nontarget soil organisms (both macro- and microorganisms). Here we review existing knowledge on key fungicides used for seed treatments, benefits and risks related to FST, and propose recommendations to increase benefits and limit risks related to the use of FST. We found FST is applied to almost 100% of sown seeds for the most important arable crops worldwide. Fungicides belonging to 10 chemical families and with one or several types of mobility (contact, locally systemic, and xylem mobile) are used for seed treatment, although the majority are xylem mobile. Seed treatments are applied by the seed distributor, the seed company, and the farmer, although the proportion of seed lots treated by these three groups vary from one crop to another. The average quantity of fungicide active ingredient (a.i.) applied via seed treatment depends on the crop species, environment(s) into which seed is planted, and regional or local regulations. Cost-effectiveness, protection of the seed and seedlings from pathogens up to 4-5 weeks from sowing, user friendliness, and lower impact on human health and nontarget soil organisms compared with foliar spray and broadcast application techniques, are among the most claimed benefits attributed to FST. In contrast, inconsistent economic benefits, development of resistance by soilborne pathogens to many fungicides, exposure risks to operators, and negative impacts on nontarget soil organisms are the key identified risks related to FST. We propose eight recommendations to reduce risks related to FST and to increase their benefits.

First report of Fusarium Wilt Tropical race 4 in Cavendish Bananas Caused by Fusarium odoratissimum in Colombia

Abstract: Fusarium wilt of bananas, commonly called Panama disease, is caused by a suite of Fusarium species. Fusarium odoratissimum (previously known as Fusarium oxysporum f.sp. cubense) comprises Tropical Race 4 (TR4) (Maryani et al., 2018), which is highly aggressive on Cavendish bananas as well as many other banana varieties (Ploetz 2015). Since the 1990's TR4 has spread across Asia until it surfaced outside this region in Jordan in 2013 (Garcia-Bastidas et al., 2014). Subsequently, a succession of TR4 incursions in banana-growing regions in Asia, the Middle East, the Indian subcontinent, Africa and even Europe was reported (Zheng et al., 2018). Thus far TR4 was not reported in Latin America. Here, we report the occurrence of TR4 in samples originating from the department of Guajira in the northeast of Colombia, which is one of the leading global banana producing countries (~550.000 ha. banana and plantain production, with 66,000 ha. for export). Typical symptoms, such as wilting and chlorosis of leaves and vascular discoloration, were observed in at least hundred plants in June 2019 in four farms totaling more than 175 ha. This area was placed under quarantine by the Colombian Agricultural Institute (ICA) and a massive eradication effort was started. In total, 22 arbitrarily pseudostem samples were collected from symptomatic Cavendish plants from two farms located 3.8 km apart. These were processed for fungal isolation and characterization as described by Zheng et al. (2018). White fungal colonies developed from surface sterilized (1% NaClO) tissue on potato dextrose agar (PDA). Three single-spore isolates (Col 2, Col 4 and Col 17) that phenotypically resembled F. odoratissimum ( Maryani et al., 2018). Subsequently, these isolates were analyzed using three molecular diagnostics targeting two independent regions of the TR4 genome. The first region was assayed according to Dita et al., (2010) and with the commercial Clear®Detections TR4 kit. The second region was tested with a Loop‐Mediated Isothermal Amplification (LAMP) assay (Ordonez et al., 2019). These tests identified the three isolates as F. odoratissimum TR4 (Figure 1). Further analyses were undertaken by whole-genome sequencing using the Illumina platform (MiSeq Kit V3) and subsequent phylogenetic analyses using the TR4 reference (Foc II5, see PRJNA73539 and PRJNA56513, GenBank) as well as available additional Fusarium spp. (Asai et al., 2019; Ordonez et al., 2015; Maryani et al., 2018; Yun et al., 2019; Zheng et al., 2018). This confirmed that the three Colombian isolates firmly group together with other F. odoratissimum TR4 isolates (Figure 1). Finally, we tested whether the three isolates were able to cause Fusarium wilt in Cavendish. The pathogenicity assays and inoculum production were according to Garcia-Bastidas et al. (2019) and included water controls, a Race 1 isolate, which is not pathogenic on Cavendish, the three Colombian isolates Col 2, Col 4, and Col 17, and the II5 TR4 reference strain. Five plants per isolate were inoculated, all plants inoculated with the Colombian TR4 isolates and the II5 reference showed typical symptoms of Fusarium wilt after four weeks. After six weeks, internal symptoms were recorded (Figure 2), and affected tissue was collected from all plants for re-isolation of the causal fungus. All affected tissues and the re-isolated strains were positive with the aforementioned TR4 diagnostics. No isolates were recovered from water controls or plants inoculated with Race 1, which remained asymptomatic (Figure 2). Taken together, our results confirm the presence of TR4 in Colombia. Despite major efforts currently undertaken to cordon off the affected farms, this is an imminent threat for Colombia and the entire region.

The Atoxigenic Biocontrol Product Aflasafe SN01 Is a Valuable Tool to Mitigate Aflatoxin Contamination of Both Maize and Groundnut Cultivated in Senegal

Abstract: Aflatoxin contamination of groundnut and maize infected by Aspergillus section Flavi fungi is common throughout Senegal. The use of biocontrol products containing atoxigenic Aspergillus flavus strains to reduce crop aflatoxin content has been successful in several regions, but no such products are available in Senegal. The biocontrol product Aflasafe SN01 was developed for use in Senegal. The four active ingredients of Aflasafe SN01 are atoxigenic A. flavus genotypes native to Senegal and distinct from active ingredients used in other biocontrol products. Efficacy tests on groundnut and maize in farmers' fields were carried out in Senegal during the course of 5 years. Active ingredients were monitored with vegetative compatibility analyses. Significant (P < 0.05) displacement of aflatoxin producers occurred in all years, districts, and crops. In addition, crops from Aflasafe SN01-treated fields contained significantly (P < 0.05) fewer aflatoxins both at harvest and after storage. Most crops from treated fields contained aflatoxin concentrations permissible in both local and international markets. Results suggest that Aflasafe SN01 is an effective tool for aflatoxin mitigation in groundnut and maize. Large-scale use of Aflasafe SN01 should provide health, trade, and economic benefits for Senegal.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Managing Colletotrichum on Fruit Crops: A "Complex" Challenge.

Abstract: The fungal genus Colletotrichum includes numerous important plant pathogenic species and species complexes that infect a wide variety of hosts. Its taxonomy is particularly complex because species' phenotypes and genotypes are difficult to differentiate. Two notable complexes, C. acutatum and C. gloeosporioides, are known for infecting temperate fruit crops worldwide. Even species within these complexes vary in traits such as tissue specificity, aggressiveness, geographic distribution, and fungicide sensitivity. With few effective chemicals available to control these pathogens, and the persistent threat of fungicide resistance, there is a need for greater understanding of this destructive genus and the methods that can be used for disease management. This review summarizes current research on diseases caused by Colletotrichum spp. on major fruit crops in the United States, focusing on the taxonomy of species involved, disease management strategies, and future management outlook.

Detection of Phytophthora infestans by Loop-Mediated Isothermal Amplification, Real-Time LAMP, and Droplet Digital PCR

Abstract: Phytophthora infestans is the causal agent of potato late blight, a devastating disease of tomato and potato and a threat to global food security. Early detection and intervention is essential for effective management of the pathogen. We developed a loop-mediated isothermal amplification (LAMP) assay for P. infestans and compared this assay to conventional PCR, real-time LAMP, and droplet digital PCR for detection of P. infestans. The LAMP assay was specific for P. infestans on potato and tomato and did not amplify other potato- or tomato-infecting Phytophthora species or other fungal and bacterial pathogens that infect potato and tomato. The detection threshold for SYBR Green LAMP and real-time LAMP read with hydroxynaphthol blue and EvaGreen was 1 pg/µl. In contrast, detection by conventional PCR was 10 pg/µl. Droplet digital PCR had the lowest detection threshold (100 fg/µl). We adapted the LAMP assay using SYBR Green and a mobile reader (mReader) for use in the field. Detection limits were 584 fg/µl for SYBR Green LAMP read on the mReader, which was more sensitive than visualization with the human eye. The mobile platform records geospatial coordinates and data from positive pathogen detections can be directly uploaded to a cloud database. Data can then be integrated into disease surveillance networks. This system will be useful for real-time detection of P. infestans and will improve the timeliness of reports into surveillance systems such as USABlight or EuroBlight.

Characterization, Phylogenetic Analyses, and Pathogenicity of Enterobacter cloacae on Rice Seedlings in Heilongjiang Province, China.

Abstract: Rice is used as a staple food in different areas of world, especially in China. In recent years, rice seedlings have been affected seriously by symptoms resembling bacterial palea browning (BPB) in Heilongjiang Province. To isolate and identify the pathogenic bacteria responsible for the disease, 40 bacterial strains were isolated from diseased rice seedlings collected from the four major accumulative-temperature zones of rice fields cultivated in Heilongjiang Province, and these were identified as 13 species based on morphological characteristics and 16S ribosomal RNA (rRNA) gene sequences. Inoculation of all the isolates on healthy rice seedlings showed that the nine Enterobacter cloacae isolates were the pathogens causing typical symptoms of BPB, including yellowing to pale browning, stunting, withering, drying, and death. Moreover, the nine E. cloacae isolates could also cause symptoms of bacterial disease on the seedlings of soybean (Glycine max), maize (Zea mays L.), and tomato (Solanum lycopersicum). Phylogenetic analysis based on the 16S rRNA gene sequences and phenotypic and biochemical characteristics indicated that these nine pathogenic isolates were E. cloacae. In addition, analysis of the sequences of four housekeeping genes (rpoB, gyrB, infB, and atpD) from the selected strain SD4L also assigned the strain to E. cloacae. Therefore, E. cloacae is the pathogen causing disease of rice seedlings in Heilongjiang Province, which we propose to classify as a form of BPB. To the best of our knowledge, this is the first study to identify E. cloacae as a causal agent of BPB in rice.

Distribution and Pathogenicity of Colletotrichum Species Associated With Mango Anthracnose in Mexico

Abstract: Mango anthracnose, caused by Colletotrichum spp., is the most significant disease of mango (Mangifera indica L.) in almost all production areas around the world. In Mexico, mango anthracnose has only been attributed to C. asianum and C. gloeosporioides. The aims of this study were to identify the Colletotrichum species associated with mango anthracnose symptoms in Mexico by phylogenetic inference using the ApMat marker, to determine the distribution of these species, and to test their pathogenicity and virulence on mango fruits. Surveys were carried out from 2010 to 2012 in 59 commercial orchards in the major mango growing states of Mexico, and a total of 118 isolates were obtained from leaves, twigs, and fruits with typical anthracnose symptoms. All isolates were tentatively identified in the C. gloeosporioides species complex based on morphological and cultural characteristics. The Bayesian inference phylogenetic tree generated with Apn2/MAT intergenic spacer sequences of 59 isolates (one per orchard) revealed that C. alienum, C. asianum, C. fructicola, C. siamense, and C. tropicale were associated with symptoms of mango anthracnose. In this study, C. alienum, C. fructicola, C. siamense, and C. tropicale are reported for the first time in association with mango tissues in Mexico. This study represents the first report of C. alienum causing mango anthracnose worldwide. The distribution of Colletotrichum species varied among the mango growing states from Mexico. Chiapas was the only state in which all five species were found. Pathogenicity tests on mango fruit cultivar Manila showed that all Colletotrichum species from this study could induce anthracnose lesions. However, differences in virulence were evident among species. C. siamense and C. asianum were the most virulent, whereas C. alienum and C. fructicola were considered the least virulent species.

ToxA–Tsn1 Interaction for Spot Blotch Susceptibility in Indian Wheat: An Example of Inverse Gene-for-Gene Relationship

Abstract: The ToxA–Tsn1 system is an example of an inverse gene-for-gene relationship. The gene ToxA encodes a host-selective toxin (HST) which functions as a necrotrophic effector and is often responsible f...

Ten Years of VINQUEST: First Insight for Breeding New Apple Cultivars With Durable Apple Scab Resistance

Abstract: Apple scab, caused by Venturia inaequalis, is a major fungal disease worldwide. Cultivation of scab-resistant cultivars would reduce the chemical footprint of apple production. However, new apple c...

First Report of Tomato Brown Rugose Fruit Virus Infecting Tomato in Greece

Abstract: During summer and autumn 2019, virus-like disease symptoms were observed in tomato (Solanum lycopersicum) grown in several greenhouses in Chania (Crete, Greece) and Kyparissia (Messenia, Peloponnese, Greece). The disease showed a particularly aggressive profile reaching in some cases 100% incidence. Symptoms included mild to severe mosaic, deformations, and blistering on leaves accompanied by brown and yellow spots on fruits. Sap inoculation of extracts from symptomatic tissues derived from seven tomato plants on Nicotiana tabacum cv. Xanthi-nc plants resulted in local necrotic lesions, indicative of a tobamovirus infection. To identify the virus, total RNAs were extracted from the seven symptomatic tomato plants and examined with a commercial tobamovirus screen reverse transcription PCR (RT-PCR) kit (LOEWE Biochemica, Germany). PCR products of 568 nucleotides were obtained from all samples examined. The seven PCR products were further subjected to sequencing. BLAST analysis of the sequences revealed the presence of tomato brown rugose fruit virus (ToBRFV) in all samples (sharing 100% nucleotide identity with each other and 99% identity with GenBank accession MK881102 from Palestine). ToBRFV, a relatively new member of the genus Tobamovirus, was originally described in Jordan (Salem et al. 2016). It is already known for its ability to overcome the so far well-established Tm-2² resistance to tobamoviruses in tomato. To further characterize the Greek isolate, total RNA from an infected tomato plant was subjected to Illumina RNA-Seq. The analysis of approximately 14,000,000, 75-bp single-end reads confirmed the presence of ToBRFV (and the absence of other viruses in the sample). BLAST analysis of the assembled sequence of the Greek isolate (ToBRFV-Gr) (6,354 nucleotides, GenBank accession no. MN815773) revealed a close relationship to the Israeli ToBRFV-IL isolate (99.8% identity, GenBank accession no. KX619418). The ToBRFV-Gr isolate was propagated through mechanical inoculation in N. tabacum ‘Turkish Samsun’, and infected tissue from the latter was used to reproduce symptoms in virus tested-negative tomato plants (cv. Rutgers). Ten days later the inoculated plants showed narrowed leaves with mild mosaic symptoms similar to those described previously by Luria et al. (2017). The presence of ToBRFV was confirmed in symptomatic leaves by RT-PCR using primer pairs ToBRFVGr-F (5′-GAATATCCGGCCTTGCAGAC-3′) and ToBRFVGr-R (5′-TCGAAATTCCACATAAGGTTGGC-3′) and by double antibody sandwich ELISA with the commercial polyclonal antibodies against ToBRFV (LOEWE Biochemica). Τhis is the first report of ToBRFV in Greece. The fact that the virus can be easily transmitted mechanically (through wounding, human activities, contact with infected plants, and insects used for pollination) and by infected seeds, in combination with the lack of any commercial resistant tomato cultivars, constitutes an emerging threat to Greek tomato and potentially pepper cultivation. ToBRFV is included in EPPO’s alert list and is regulated in the European Union since November 2019 (Commission Implementing Decision [EU] 2019/1615). Containment actions against the further spread of ToBRFV were taken at the sites of its emergence.

Trichoderma Isolates Inhibit Fusarium virguliforme Growth, Reduce Root Rot, and Induce Defense-Related Genes on Soybean Seedlings.

Abstract: Sudden death syndrome (SDS) caused by Fusarium virguliforme is among the most important diseases affecting soybean in the United States. The use of biological control agents (BCAs) such as Trichoderma spp. can be a valuable resource to suppress F. virguliforme populations. Therefore, this research focused on screening possible BCAs against F. virguliforme and evaluating mycoparasitism and the induction of systemic resistance as mechanisms underlying the antagonistic activity of selected BCAs against F. virguliforme. In total, 47 potential BCAs, including 41 Trichoderma isolates and 6 Mortierella isolates, were screened in a dual-plate assay. The most effective isolates belonged to the Trichoderma harzianum species and were able to inhibit F. virguliforme radial growth by up to 92%. Selected Trichoderma isolates were tested in the greenhouse and in a microplot study. They reduced root rot caused by F. virguliforme when the plants were coinoculated with the pathogen and the BCA. The tested BCA's ability to reduce F. virguliforme growth may be related to several mechanisms of action, including mycoparasitism and induction of defense-related genes in plants, as revealed by monitoring the expression of defense-related genes in soybean. Our results highlight the potential of native Trichoderma isolates to inhibit F. virguliforme growth and reduce SDS severity, providing the basis for future implementation of biological control in soybean production. More efforts are needed to implement the use of these approaches in production fields, and to deepen the current knowledge on the biology of these highly antagonistic isolates.

Novel Sources of Wheat Head Blast Resistance in Modern Breeding Lines and Wheat Wild Relatives.

Abstract: Wheat head blast (WHB), caused by the fungus Magnaporthe oryzae pathotype triticum, is a devastating disease affecting South America and South Asia. Despite 30 years of intensive effort, the 2NVS translocation from Aegilops ventricosa contains the only useful source of resistance to WHB effective against M. oryzae triticum isolates. The objective of this study was to identify non-2NVS sources of resistance to WHB among elite cultivars, breeding lines, landraces, and wild-relative accessions. Over 780 accessions were evaluated under field and greenhouse conditions in Bolivia, greenhouse conditions in Brazil, and at two biosafety level-3 laboratories in the United States. The M. oryzae triticum isolates B-71 (2012), 008 (2015), and 16MoT001 (2016) were used for controlled experiments, while isolate 008 was used for field experiments. Resistant and susceptible checks were included in all experiments. Under field conditions, susceptible spreaders were inoculated at the tillering stage to guarantee sufficient inoculum. Disease incidence and severity were evaluated as the average rating for each 1-m-row plot. Under controlled conditions, heads were inoculated after full emergence and individually rated for percentage of diseased spikelets. The diagnostic marker Ventriup-LN2 was used to test for the presence of the 2NVS translocation. Four non-2NVS spring wheat International Maize and Wheat Improvement Center breeding lines (CM22, CM49, CM52, and CM61) and four wheat wild-relatives (A. tauschii TA10142, TA1624, TA1667, and TA10140) were identified as resistant (<5% of severity) or moderately resistant (5 to <25% severity) to WHB. Experiments conducted at the seedling stage showed little correlation with disease severity at the head stage. M. oryzae triticum isolate 16MoT001 was significantly more aggressive against 2NVS-based varieties. The low frequency of WHB resistance and the increase in aggressiveness of newer M. oryzae triticum isolates highlight the threat that the disease poses to wheat production worldwide and the urgent need to identify and characterize new resistance genes that can be used in breeding for durably resistant varieties.

Characterization and Fungicide Sensitivity of Colletotrichum Species Causing Strawberry Anthracnose in Eastern China.

Abstract: Strawberry anthracnose caused by Colletotrichum spp. is one of the most serious diseases in the strawberry fields of China. In total, 196 isolates of Colletotrichum were obtained from leaves, stolons, and crowns of strawberry plants with anthracnose symptoms in eastern China and were characterized based on morphology, internal transcribed spacer (ITS), and β-tubulin (TUB2) gene sequences. All 196 isolates were identified as the Colletotrichum gloeosporioides species complex. In total, 62 strains were further identified at the species level by phylogenetic analyses of multilocus sequences of ITS, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat), calmodulin (CAL), and TUB2. Three species from the C. gloeosporioides species complex were identified: Colletotrichum siamense, C. fructicola, and C. aenigma. Isolates of C. siamense were tolerant to high temperatures, with a significantly larger colony diameter than the other two species when grown above 36°C. The inoculation of strawberry plants confirmed the pathogenicity of all three species. C. siamense isolates resulted in the highest disease severity. The in vitro sensitivities of C. siamense and C. fructicola isolates to azoxystrobin and three demethylation-inhibitor (DMI) fungicides (difenoconazole, tebuconazole, and prochloraz) were determined. Both species were sensitive to DMI fungicides but not to azoxystrobin. C. siamense isolates were more sensitive to prochloraz, while C. fructicola isolates were more sensitive to difenoconazole and tebuconazole. The present study provides valuable information for the effective management of strawberry anthracnose.

Enhancing PCR Capacity To Detect 'Candidatus Liberibacter asiaticus' Utilizing Whole Genome Sequence Information.

Abstract: 'Candidatus Liberibacter asiaticus' (CLas) is an unculturable α-proteobacterium associated with citrus Huanglongbing (HLB; yellow shoot disease). PCR procedures that accurately confirm or exclude CLas infection in citrus tissue/Asian citrus psyllid (ACP) samples are critical for HLB management. When CLas was described in 1994, a 23-bp signature oligonucleotide sequence (OI1) in the 16S rRNA gene (rrs, three genomic copies) was identified based on Sanger sequencing. OI1 contains single nucleotide polymorphisms (SNPs) distinguishing CLas from non-CLas species. The SNPs were used to design the primer HLBas, a key primer for a commonly used TaqMan PCR system (HLBas-PCR) for CLas detection. Recent developments in next-generation sequencing technology have led to the identification of 15 CLas whole genome sequence strains (WGSs). Analyses of CLas WGSs have generated a significant amount of biological information that could help to improve CLas detection. Utilizing the WGS information, this study re-evaluated the sequence integrity of OI1/HLBas and identified and/or confirmed a missing nucleotide G in the two primers. Replacement primers for OI1 and HLBas are proposed. At low cycle threshold (Ct) values (e.g., 30), HLBas-PCR alone was unreliable in differentiating whether samples contain low CLas titers or whether CLas is not present. The availability of ribonucleotide reductase (RNR)-PCR derived from the five-copy nrdB gene helped to resolve this problem. To further enhance low CLas titer detection, a 4CP-PCR system, based on a four-copy genomic locus, was developed. Evaluation of 107 HLB samples (94 citrus and 13 ACP) showed that 4CP-PCR was more sensitive than HLBas-PCR and shared similar sensitivity with RNR-PCR.

Mutations and Overexpression of CYP51 Associated with DMI-Resistance in Colletotrichum gloeosporioides from Chili.

Abstract: Chili anthracnose caused by Colletotrichum spp. is an annual production concern for growers in China. Sterol C14-demethylation inhibitors (DMIs, such as tebuconazole) have been widely used to control this disease for more than three decades. In the current study, of 48 isolates collected from commercial chili farms in Jiangsu Province of China during 2018 and 2019, 8 single-spore isolates were identified as Colletotrichum gloeosporioides and the rest were identified as C. acutatum. To determine whether the DMI resistance of isolates develops in the field, mycelial growth of the 48 isolates was measured in culture medium with and without tebuconazole. In all, 6 of the 8 C. gloeosporioides isolates were resistant to tebuconazole, but all 40 of the C. acutatum isolates were sensitive to tebuconazole. The fitness cost of resistance was low based on a comparison of fitness parameters between the sensitive and resistant isolates of C. gloeosporioides. Positive cross-resistance was observed between tebuconazole and difenconazole or propiconazole, but not prochloraz. Alignment results of the CgCYP51 amino acid sequences from the sensitive and resistant isolates indicated that mutations can be divided into three genotypes. Genotype I possessed four substitutions (V18F, L58V, S175P, and P341A) at the CgCYP51A gene but no substitutions at CgCYP51B, while genotype II had five substitutions (L58V, S175P, A340S, T379A, and N476T) at CgCYP51A, concomitant with three substitutions (D121N, T132A, and F391Y) at CgCYP51B. In addition, genotype III contained two substitutions (L58V and S175P) at CgCYP51A, concomitant with one substitution (T262A) at CgCYP51B. Molecular docking models illustrated that the affinity of tebuconazole to the binding site of the CgCYP51 protein from the resistant isolates was decreased when compared with binding site affinity of the sensitive isolates. Our findings provide not only novel insights into understanding the resistance mechanism to DMIs, but also some important references for resistance management of C. gloeosporioides on chili.

First Inventory of Fungi in Symptomless and Symptomatic Chinese Mesona Indicates Phytopathological Threat.

Abstract: Chinese mesona (Platostoma palustre) plays an important role as special crop in Southeast Asia and Taiwan for the production of herbal tea, grass jelly, and further processed food. In order to assess the potential threat of fungi to Chinese mesona, we surveyed isolates from symptomless plants in the area of mesona production, as well as from leaf spots of potted plants in a garden shop and a plantation in a botanical garden in Taiwan. From leaves, stems, and roots of 15 symptomless plants sampled at five collection events over two years, 154 isolates from 810 surface-sterilized plant fragments were obtained and identified based on DNA sequence data of the internal transcribed spacer region, and partially of the β-tubulin and histone H3 genes. The most common species belonged to the genera Cercospora, Colletotrichum, and Fusarium and were considered to be potential plant pathogens. Latent pathogenicity was confirmed by an infection experiment with an endophytic strain of Corynespora cassiicola. Observation of leaf spot disease associated with Cercospora kikuchii suggested pathogenicity of this fungus, which was also isolated as an endophyte from symptomless leaves. We hypothesize that the most common endophytic fungi are latent pathogens in the host and may cause plant disease when the host becomes weakened by senescence or changed cultivation condition. Leaf spots of plants in the botanical garden were associated with a species of Pseudocercospora, which was not found among the endophytic isolates and is newly described based on morphology and analysis of translation elongation factor 1 alpha gene sequences.

Occurrence and diversity of black-foot disease fungi in symptomless grapevine nursery stock in Spain.

Abstract: In this study, 3,426 grafted grapevines ready to be planted from 15 grapevine nursery fields in Northern Spain were inspected from 2016 to 2018 for black-foot causing pathogens. In all, 1,427 isolates of black-foot pathogens were collected from the asymptomatic inner tissues of surface sterilized secondary roots and characterized based on morphological features and DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, histone H3, translation elongation factor 1-alpha and β-tubulin genes. Eleven species belonging to the genera Dactylonectria, Ilyonectria, Neonectria, and Thelonectria were identified, including Dactylonectria alcacerensis, D. macrodidyma, D. novozelandica, D. pauciseptata, D. torresensis, Ilyonectria liriodendri, I. pseudodestructans, I. robusta, Neonectria quercicola, Neonectria sp. 1, and Thelonectria olida. In addition, two species are newly described, namely D. riojana and I. vivaria. Twenty-four isolates representing 13 black-foot species were inoculated onto grapevine seedlings cultivar 'Tempranillo'. The pathogenicity tests detected diversity in virulence among fungal species and between isolates within each species. The most virulent species was D. novozelandica isolate BV-0760, followed by D. alcacerensis isolate BV-1240 and I. vivaria sp. nov. isolate BV-2305. This study improves our knowledge on the etiology and virulence of black-foot disease pathogens, and opens up new perspectives in the study of the endophytic phase of these pathogens in grapevines.

First report of tobacco mild green mosaic virus and tomato brown rugose fruit virus infecting Capsicum annuum in Jordan.

Abstract: Pepper (Capsicum annuum L.) is one of the major vegetable crops in Jordan. During the winter growing seasons of 2015 and 2016, virus symptoms including stunting of young plants, puckering, and yellow mottling of leaves in sweet pepper plants grown under plastic houses in Jordan Valley were observed. The most obvious symptoms were the misshapen fruits that affected the market value of the crop, which resembled recent descriptions of tomato brown rugose fruit virus (ToBRFV) on tomato fruits (Salem et al. 2016). Symptoms on mechanically inoculated indicator hosts including Chenopodium quinoa, Datura stramonium, D. metel, Nicotiana glutinosa, and N. tabacum, and serological testing using double-antibody sandwich ELISA (antiserum A128 of the PLAVIT collection at IPSP-CNR, Italy) suggested the presence of a tobamovirus. Total RNA was extracted from fruits and leaves of 39 symptomatic pepper plants, using an SV-Total RNA Extraction kit (Promega, U.S.A.). Samples were tested by reverse transcription polymerase chain reaction (RT-PCR) for the most common tobamoviruses infecting pepper, including tomato mosaic virus, tobacco mosaic virus, tobacco mild green mosaic virus (TMGMV), and pepper mild mottle virus (Takeuchi et al. 2005). In addition, generic primers for detection of tobamoviruses were also used (Dovas et al. 2004). In RT-PCR, amplicons of the expected product size (400 and 728 bp) using the generic tobamovirus and TMGMV-specific primers, respectively, in all symptomatic plant samples were obtained, but such amplicons were not obtained from healthy plant extracts or water negative controls. RT-PCR products of the RdRp (400 bp) and CP (728 bp) partial regions were purified and ligated into pGEM T-Easy Vector (Promega), and two clones for each PCR product were sequenced and deposited in NCBI GenBank (accession nos. MK816313 to 16). BLASTN analysis showed that these nucleotide sequences had 96 to 99% identity to TMGMV genome sequences (JX534224 and MH730962) in NCBI GenBank. Furthermore, total RNA was extracted with TRIzol reagent (Invitrogen, U.S.A.) according to the manufacturer’s specifications. After ribosomal RNA depletion, the cDNA library was constructed using a TruSeq RNA Sample Prep kit (Illumina, U.S.A.) and sequenced by Illumina HiSeq X-ten platform (Biomarker, China). Raw sequencing data were analyzed using CLC Genomics Workbench 9.5 (Qiagen, Denmark). After raw reads were processed, a total of 80,818,734 paired-end reads of 150 bp were obtained, generating 266,412 contigs (>200 nt) with de novo assembly by CLC Genomics Workbench 9.5. BLASTN analysis of the assembled contigs revealed the presence of two virus-derived contigs: TMGMV (6,414 nt, MK648158) and ToBRFV (6,388 nt, MK648157), which represented a nearly full-length genome. To confirm the ToBRFV identity, all samples were tested by RT-PCR using two pairs of primers: ToBRFV F1 (5′-GTATTTTTGTTTTACAACATATACCAAC-3′) and ToBRFV R1 (5′-AGTGCGAATGTGATTTAAAACTGTGAA-3′), and ToBRFV F7 (5′-GGAAGAAGTCCCGATGTCTGTAAGGCTT-3′) and ToBRFV R7 (5′-GATGCAGGTGCAGAGGACCATTGTAAAC-3′), designed on ToBRFV genome (KT383474; Salem et al. 2016). Twenty-two out of 39 tested samples originated specific amplicons (1,300 and 697 bp for RdRp and CP, respectively). RT-PCR products of two samples were purified and sent for direct sequencing, and results were deposited in NCBI GenBank (MK834288, MK834289, MK834294, and MK834295), revealing nucleotide identity of 99 to 100% to ToBRFV for both RdRp and CP (KT383474 and KX619418). To our knowledge, this is the first report of TMGMV and ToBRFV infecting pepper in Jordan. The occurrence of these tobamoviruses that are transmitted through seeds, in Jordan Valley, the main area for pepper production, may represent a potential threat to other susceptible vegetables and requires careful monitoring to avoid future outbreaks and significant yield losses.

Cover Crop Rotation Effects on Growth and Development, Seedling Disease, and Yield of Corn and Soybean

Abstract: The effects of winter cover crops on root disease and growth of corn and soybeans are poorly understood. A 3-year field experiment investigated the effect of winter cereal rye (Secale cereale L.) and winter camelina (Camelina sativa [L.] Crantz), used either in all three years or in rotation with each other, on corn (Zea mays L.) and soybean (Glycine max. [L.] Merr.) growth, root disease, and yield. Corn following a cover crop of camelina had reduced root disease, a lower Pythium population in seedling roots, and greater growth and yields compared with corn following a rye cover crop. Camelina and rye cover crops before soybean had either a positive or no effect on soybean growth and development, root disease, and yield. Moreover, Pythium clade B populations were greater in corn seedlings after a rye cover crop compared with those following a camelina cover crop, whereas clade F populations were greater on soybean seedlings following a camelina cover crop compared with seedlings following a rye cover crop. A winter camelina cover crop grown before corn had less-negative effects on corn seedling growth, root disease, and final yield than a winter rye cover crop before corn. Neither cover crop had negative effects on soybean, and the cover crop in the preceding spring had no measurable effects on either corn or soybean.

Genetic Diversity of Colletotrichum spp. Causing Strawberry Anthracnose in Zhejiang, China

Abstract: Anthracnose is a serious fungal disease that primarily infects strawberry roots and stolons during development. Here, 91 isolates from different areas of Zhejiang province, China, were collected. M...

New North American Isolates of Venturia inaequalis Can Overcome Apple Scab Resistance of Malus floribunda 821.

Abstract: Apple scab, caused by Venturia inaequalis, is a destructive fungal disease of major apple cultivars worldwide, most of which are moderately to highly susceptible. Thus, development of scab resistan...

Etiology of Branch Dieback and Shoot Blight of English Walnut Caused by Botryosphaeriaceae and Diaporthe Species in Southern Spain.

Abstract: English walnut (Juglans regia L.) is considered an economically important fruit crop worldwide. In Spain, little attention has been given to walnut diseases owing to the minor economic importance of the walnut crop in the country until recently. In 2017, typical symptoms of branch dieback and shoot blight of English walnut were observed in southern Spain. From 2017 to 2018, 10 commercial walnut orchards showing disease symptoms were surveyed. Botryosphaeriaceae and Diaporthe fungi were consistently isolated from affected shoots. Cytospora isolates were also recovered with minor relevance. Representative isolates of each fungal group were characterized based on colony and conidial morphology, optimum growth temperature, and comparison of DNA sequence data from the internal transcribed spacer, elongation factor 1-α, and β-tubulin genomic areas. Pathogenicity tests were performed on detached and attached shoots and on detached fruit by inoculating them with mycelial plugs. Botryosphaeriaceae and Diaporthe isolates had higher optimum growth temperatures (≈25 to 27°C) than Cytospora sp. (19.5°C). The following species were identified: Botryosphaeriaceae: Botryosphaeria dothidea, Diplodia seriata, Dothiorella sarmentorum, Dothiorella sp., Neofusicoccum mediterraneum, and N. parvum; Diaporthe: Diaporthe neotheicola, Dia. rhusicola, Diaporthe sp., and Phomopsis amygdali; and Cytospora sp. Botryosphaeriaceae isolates were the most aggressive fungi to walnut in all tissues evaluated, followed by Diaporthe isolates and Cytospora sp. N. parvum was the most virulent among the remaining species tested in any of the tissues evaluated, followed by B. dothidea or N. mediterraneum. This work is the first report to identify the fungal species causing this complex disease of English walnut in Spain and Europe.

Pathogenicity, Symptom Development, and Colonization of Metrosideros polymorpha by Ceratocystis lukuohia.

Abstract: Extensive mortality of Metrosideros polymorpha (`ōhi`a) trees has been associated with Ceratocystis spp. on Hawai`i Island and was named rapid `ōhi`a death (ROD). Both C. lukuohia and C. huliohia h...

Identification and Molecular Mapping of a Gummy Stem Blight Resistance Gene in Wild Watermelon (Citrullus amarus) Germplasm PI 189225.

Abstract: Gummy stem blight (GSB), caused by Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), is a destructive foliar disease of watermelon in areas with hot and humid climates. The wild watermelon germplasm PI 189225 is a known source of resistance to GSB. The identification and use of molecular markers linked to resistance genes in the wild-type germplasm will speed up the introgression of GSB resistance into new watermelon varieties. An F2 segregating population was obtained from a cross between the resistant wild watermelon genotype PI 189225 and the susceptible genotype K3. The F2-derived F3 families were inoculated with a single isolate of S. cucurbitacearum (JS002) from Jiangsu Academy of Agricultural Sciences. The results of the genetic analysis demonstrated that GSB resistance in PI 189225 was controlled by a major quantitative trait locus (QTL), temporarily designated Qgsb8.1. Based on the results of bulk sergeant analysis and sequencing, one associated region spanning 5.7 Mb (10,358,659 to 16,101,517) on chromosome 8 was identified as responsible for the resistance to GSB using the Δ(single-nucleotide polymorphism [SNP]-index) method. The result of a QTL linkage analysis with Kompetitive allele-specific PCR (KASP) SNP markers further mapped the GSB resistance locus between the SNP markers KASP_JS9383 and KASP_JS9168 in a region of 571.27 kb on chromosome 8. According to the watermelon gene annotation database, the region contains approximately 19 annotated genes and, of these 19 genes, 2 are disease resistance gene analogs: Cla001017 (coiled-coil nucleotide-binding site leucine-rich repeat resistance protein) and Cla001019 (pathogenesis related). Reverse-transcription quantitative PCR demonstrated that the expression of the two genes changed following S. cucurbitacearum infection, suggesting that they play important roles in GSB resistance in watermelon. This result will facilitate fine mapping and cloning of the Qgsb8.1 locus, and the linked markers will further provide a useful tool for marker-assisted selection of this locus in watermelon breeding programs.

Identification, Virulence and Fungicide Sensitivity of Colletotrichum gloeosporioides s.s. Responsible for Walnut Anthracnose Disease in China.

Abstract: Walnut (Juglans regia L.) is an economically important woody nut and edible oil tree all over the world. However, walnut production is limited by walnut anthracnose, which is a disastrous disease that causes significant yield losses. Studying the etiology of anthracnose on walnut and the pathogens' virulence and sensitivities to fungicides would be beneficial for effective control. This study was conducted to identify the pathogen of walnut anthracnose and reveal the population diversity of pathogens through virulence, sensitivities to fungicides, and genetic variation. A total of 13 single-spore Colletotrichum isolates were collected from walnut anthracnose-diseased fruits and leaves from 13 walnut commercial orchards in Henan, Hubei, Shandong, and Shaanxi provinces in China. The isolates were identified as Colletotrichum gloeosporioides sensu stricto (s.s.) according to multilocus phylogenetic analyses (internal transcribed spacer, actin, glyceraldehyde-3-phosphate dehydrogenase, and chitin synthase), morphological as well as cultural characters, and pathogenicity. When the same walnut tissue was inoculated with different isolates, the disease lesion size was different. The results showed that the virulence of all isolates was considerably different, and the differences were not correlated with geographic origins. The virulence to walnut leaves and fruits inoculated with the same isolate was significantly different. Based on the virulence to walnut leaves and fruits, the 13 isolates were divided into three groups. Virulence of 69.2% of the isolates to walnut fruits was higher than that to leaves; 15.4% of isolates had no difference in pathogenicity, and the virulence to walnut leaves was higher for 15.4% of isolates. Tebuconazole, difenoconazole, flusilazole, and carbendazim inhibited the growth of fungal mycelia, and the concentration for 50% of maximal effect (EC50) values were 0.4 to 20.5, 0.6 to 2.6, 0.2 to 1.6, and 0.002 to 0.2 µg/ml, respectively, with average values of 6.5 ± 6.9, 1.5 ± 0.6, 0.9 ± 0.4, and 0.1 ± 0.05 µg/ml, respectively. All isolates were more sensitive to difenoconazole, flusilazole, and carbendazim than tebuconazole (P < 0.01). Isolate sensitivities to the same fungicide were different. Isolates SL-31 and TS-09 were the least sensitive to carbendazim and tebuconazole, respectively, and the resistance ratios were 87.3 and 51.6, respectively. Sensitivities to difenoconazole and flusilazole were largely consistent among all isolates, and the resistance ratios were from 1 to 4.6 and from 1 to 7, respectively. Therefore, difenoconazole and flusilazole could be chosen for disease control. The differences of pathogenicity and fungicide sensitivity were not correlated with geographic regions. These results indicated that there was high intraspecific diversity of populations in C. gloeosporioides s.s. that caused walnut anthracnose. For effective management, the targeted control strategy should be implemented based on the different geographic regions.

Identification of a Recessive Gene PmQ Conferring Resistance to Powdery Mildew in Wheat Landrace Qingxinmai Using BSR-Seq Analysis.

Abstract: Wheat powdery mildew is caused by Blumeria graminis f. sp. tritici (Bgt), a biotrophic fungal species. It is very important to mine new powdery mildew (Pm) resistance genes for developing resistant wheat cultivars to reduce the deleterious effects of the disease. This study was carried out to characterize the Pm gene in Qingxinmai, a winter wheat landrace from Xinjiang, China. Qingxinmai is resistant to many Bgt isolates collected from different wheat fields in China. F1, F2, and F2:3 generations of the cross between Qingxinmai and powdery mildew susceptible line 041133 were developed. It was confirmed that a single recessive gene, PmQ, conferred the seedling resistance to a Bgt isolate in Qingxinmai. Bulked segregant analysis-RNA-Seq (BSR-Seq) was performed on the bulked homozygous resistant and susceptible F2:3 families, which detected 57 single nucleotide polymorphism (SNP) variants that were enriched in a 40 Mb genomic interval on chromosome arm 2BL. Based on the flanking sequences of the candidate SNPs extracted from the Chinese Spring reference genome, 485 simple sequence repeat (SSR) markers were designed. Six polymorphic SSR markers, together with nine markers that were anchored on chromosome arm 2BL, were used to construct a genetic linkage map for PmQ. This gene was placed in a 1.4 cM genetic interval between markers Xicsq405 and WGGBH913 corresponding to 4.9 Mb physical region in the Chinese Spring reference genome. PmQ differed from most of the other Pm genes identified on chromosome arm 2BL based on its position and/or origin. However, this gene and Pm63 from an Iranian common wheat landrace were located in a similar genomic region, so they may be allelic.

Exogenous Melatonin Enhances Rice Plant Resistance Against Xanthomonas oryzae pv. oryzae

Abstract: Rice bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae, is one of the most serious diseases of rice. In this study we found that exogenous melatonin can increase rice resistance to BB. Treatment of rice plants with exogenous melatonin (20 µg/ml) increased nitrate reductase, nitric oxide synthase, and peroxidase activity, enabling high intracellular concentrations of melatonin, nitric oxide, and H2O2. The expression of NPR1, a key regulator in the salicylic acid signaling pathway, was upregulated more than 10-fold when the plants were challenged with melatonin. Similarly, the messenger RNA level of PDF1.2, a jasmonic acid-induced defense marker, was 15 times higher in the treated plants than in the control plants. Moreover, three pathogenesis-related proteins, PR1b, PR8a, and PR9, were upregulated 20-fold in the presence of melatonin. The application of melatonin (100 µg/ml) to soil-grown rice reduced the incidence of BB by 86.21%. Taken together, these results not only provide a better understanding of melatonin-mediated innate immunity to X. oryzae pv. oryzae in rice but also represent a promising cultivation strategy to protect rice against X. oryzae pv. oryzae infection.

Characteristics and Rapid Diagnosis of Pectobacterium carotovorum ssp. Associated With Bacterial Soft Rot of Vegetables in China.

Abstract: Pectobacterium carotovorum, a causal agent of vegetable soft rot, contains three valid subspecies: P. carotovorum subsp. carotovorum (Pcc), P. carotovorum subsp. brasiliensis (Pcb), and P. carotovorum subsp. odoriferum (Pco). Using 16S rDNA sequencing and genus-specific PCR, we identified 72 P. carotovorum strains from Chinese cabbage, bok choy, and celery and assessed their pathogenicity on Chinese cabbage petioles and potato tubers. Based on phylogenetic analysis of pmrA sequences and confirmation by subspecies-specific PCR, the strains were divided into 18 Pcc, 29 Pco, and 25 Pcb. Several characteristic features were also assessed and supported the distinctiveness of the Pco strains. All P. carotovorum strains caused soft rot symptoms on Chinese cabbage and potato, but the Pco strains exhibited the greatest severity. We developed a conventional PCR and a quantitative PCR (qPCR) assay for the identification of Pco based on its specific srlE gene encoding sorbitol-specific phosphotransferase. These two methods could specifically amplify the expected products of 674 and 108 bp, respectively, from all of the Pco strains. The assays demonstrated high sensitivity and could detect as little as 1 and 100 pg/µl of bacterial genomic DNA, respectively. Both assays could also detect the pathogens directly from plant tissues infected with as little as 2.5 × 10-2 CFU/mg of Pco, even before external symptoms appeared. These assays constitute effective tools for disease diagnosis and the rapid identification of soft rot pathogens.

Efficacy of Natamycin Against Gray Mold of Stored Mandarin Fruit Caused by Isolates of Botrytis cinerea With Multiple Fungicide Resistance.

Abstract: Gray mold caused by Botrytis cinerea is an emerging postharvest disease of mandarin fruit in California. Management of postharvest diseases of mandarins relies on postharvest fungicides; however, multiple resistance to fungicides of different modes of action is common in B. cinerea populations from mandarin, leading to their failure to control decay. Natamycin is commonly used in the food industry as an additive, and it has been registered as a biofungicide for postharvest use on citrus and some other fruits. Sensitivity to natamycin of 64 isolates of B. cinerea from decayed mandarin fruit with known resistance phenotypes to other citrus postharvest fungicides (azoxystrobin, fludioxonil, pyrimethanil, and thiabendazole) was tested. Effective concentrations of natamycin to cause a 50% reduction relative to the control for conidial germination were from 0.324 to 0.567 µg/ml (mean of 0.444 µg/ml), and those for mycelial growth were 1.021 to 2.007 µg/ml (mean of 1.578 µg/ml). Minimum inhibitory concentrations where no fungal growth was present were 0.7 to 1.0 µg/ml for conidial germination and 5.0 to 10.0 µg/ml for mycelial growth. No cross-resistance between natamycin and other citrus postharvest fungicides was detected. Decay control efficacy tests with natamycin were conducted on mandarin fruit inoculated with B. cinerea isolates exhibiting five different fungicide resistance phenotypes, and natamycin significantly reduced incidence and lesion size of gray mold on fruit, regardless of fungicide resistance phenotypes. Natamycin has the potential to be an effective tool for integration into postharvest fungicide programs to control gray mold and manage B. cinerea isolates resistant to fungicides with other modes of action.