Whole-plant Assays Research Articles

First report of anthracnose caused by Colletotrichum fructicola on winter jasmine (Jasminum nudiflorum) in China.

Winter jasmine (Jasminum nudiflorum Lindl.) is a medium-sized, deciduous shrub native to China that has become a popular choice among gardeners and landscapers. In 2020 to 2021, symptoms of anthracnose including brown necrotic spots, enlarged irregular lesions and leaf blight were observed on leaves of 20 winter jasmine shrubs in a public garden (22°34'58'' N; 113°56'23'' E) in Shenzhen, China, and with an estimated disease incidence of 65%. Tissues samples (6 × 6 mm2) surrounding the necrotic spots were surface sterilized with 75% ethanol for 30 s, followed by 2% NaClO for 1 min, then rinsed with sterile water for three times and dried with sterile filter paper. Tissues were placed on potato dextrose agar (PDA) medium and incubated at 25℃. After 3 to 7 d, pure cultures were obtained by transferring hyphal tips to new plates and 32 isolates producing Colletotrichum-like colonies were obtained from 40 tissues (isolation frequency=32/(4×10)=80%). Three representative isolates YCH09, YCH23 and YCH32 were selected for further study. Three selected isolates were identical in morphological characteristics. Colonies on PDA after 5 d at 25℃ were white to gray with cottony mycelia and grayish-white on the underside of the culture. Conidia (n = 60) measured 15.4 ± 1.1 µm (13.0 to 17.1 µm) in length and 5.4 ± 0.3 µm (4.9 to 6.0 µm) in width and were hyaline, single-celled, cylindrical with rounded ends. Appressoria (n = 15) measured 7.1 ± 0.1 µm (5.3 to 8.9 µm) in length and 5.2 ± 0.2 µm (4.1 to 6.2 µm) in width and were brown to dark brown, ovoid. These morphological features were aligned with those of Colletotrichum spp. (Weir et al. 2012). Sequences of five genetic markers of representative isolates YCH09, YCH23 and YCH32 including the rDNA internal transcribed spacer region, chitin synthase, partial actin, β-tubulin 2 and Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) region were 99.3 to 100% identical to the ex-type isolate of C. fructicola strain ICMP 18581 (Zhang et al., 2020). From the maximum likelihood phylogenetic tree which was constructed based on concatenated sequences, three representative isolates (YCH09, YCH23 and YCH32) were clustered with other isolates of C. fructicola. The above morphological and molecular characteristics suggest that causal agent was C. fructicola. Pathogenicity was tested using a whole-plant assay. Five healthy plants were inoculated by spraying a conidial suspension (1.5×104 conidia/ml; 20 ml per plant) of the isolate YCH23 onto the foliage (Marshall et al., 2023). Three noninoculated control plants were sprayed with sterile water. All plants were placed in a greenhouse at 25±2℃ with approximately 75% relative humidity. Yellow lesions appeared on leaves of inoculated plants as early as 4 days after inoculation (DAI), and irregularly shaped brown spots similar to those observed in the field were formed on 10 DAI. Noninoculated plants remained asymptomatic. Colletotrichum isolates resembling morphological characters of YCH23 were reisolated from all inoculated plants, then identified as C. fructicola by DNA sequence analysis. C. fructicola is a well-known fungus causing anthracnose on more than 63 plant species including agricultural and horticultural plants worldwide (Talhinhas and Baroncelli, 2021). To our knowledge, this is the first report of C. fructicola infecting J. nudiflorum plants in China. Since its potential risk to other horticultural plant species, precautions may be necessary to minimize the spread of this fungi.

Pathogenicity of Single and Combined Inoculations of Alternaria spp. on Potato

AbstractIn Europe, early blight has become an increasingly severe disease in potato cultivation. We investigated the lesion sizes caused by infection with different Alternaria spp. predominantly from the UK in detached-leaf assays and whole-plant assays of different potato cultivars. The analysis reveals that whilst there is no significant interaction between the potato cultivars used and the Alternaria spp. tested, statistically significant differences were observed between the group means of small-spored vs. large-spored Alternaria spp. The small-spored species grouped together with no significant differences amongst them, whilst large-spored species were able to cause large expanding lesions in all tests, with Alternaria solani exhibiting the most extensive lesion growth. We also explored the effect of co-inoculation treatments of Alternaria spp., revealing no significant difference between the inoculation with Alternaria solani alone and co-inoculation with Alternaria solani and Alternaria alternata or Alternaria infectoria. The findings contribute to our understanding of the behaviour of Alternaria spp. and their impact on potato cultivars.

Exogenous double-stranded RNA inhibits the infection physiology of rust fungi to reduce symptoms in planta.

Rust fungi (Pucciniales) are a diverse group of plant pathogens in natural and agricultural systems. They pose ongoing threats to the diversity of native flora and cause annual crop yield losses. Agricultural rusts are predominantly managed with fungicides and breeding for resistance, but new control strategies are needed on non-agricultural plants and in fragile ecosystems. RNA interference (RNAi) induced by exogenous double-stranded RNA (dsRNA) has promise as a sustainable approach for managing plant-pathogenic fungi, including rust fungi. We investigated the mechanisms and impact of exogenous dsRNA on rust fungi through in vitro and whole-plant assays using two species as models, Austropuccinia psidii (the cause of myrtle rust) and Coleosporium plumeriae (the cause of frangipani rust). In vitro, dsRNA either associates externally or is internalized by urediniospores during the early stages of germination. The impact of dsRNA on rust infection architecture was examined on artificial leaf surfaces. dsRNA targeting predicted essential genes significantly reduced germination and inhibited development of infection structures, namely appressoria and penetration pegs. Exogenous dsRNA sprayed onto 1-year-old trees significantly reduced myrtle rust symptoms. Furthermore, we used comparative genomics to assess the wide-scale amenability of dsRNA to control rust fungi. We sequenced genomes of six species of rust fungi, including three new families (Araucariomyceaceae, Phragmidiaceae, and Skierkaceae) and identified key genes of the RNAi pathway across 15 species in eight families of Pucciniales. Together, these findings indicate that dsRNA targeting essential genes has potential for broad-use management of rust fungi across natural and agricultural systems.

First report of Alternaria cinerariae causing leaf blight on Farfugium japonicum in South Carolina, USA.

Farfugium japonicum, commonly known as leopard plant, is a popular perennial used in landscapes in the Southeastern U.S. In March 2022, leaf blight was observed on 20 leopard plants at a landscape site in Georgetown Co., SC. Almost all leaves were infected. Symptoms included purple to brown necrotic leaf spots and blighted petioles. Large spots had concentric circles and coalesced causing entire leaves to blight. Leaf pieces surrounding necrotic spots were excised, sterilized in 10% bleach for 1 min, rinsed in sterile water, placed onto potato dextrose agar (PDA), and incubated at 25°C. A total of three Alternaria isolates, 22-094-A, 22-094-B, and 22-094-C were obtained by transferring hyphal tips to new plates. All isolates had identical morphological traits. Colonies on PDA were blackish at the center and brownish at the edge. Conidia were produced using a technique described by Shahin and Shepard (1979). Conidiophores were mostly short and unbranched. They were characterized by solitary conidia or short chains of two to three conidia. Conidia (n=30) were obpyriform to obclavate and averaged 88.5 ± 26.1 µm in body length, 118.4 ± 36.3 µm in total length, and 23.9 ± 5.9 µm in width. They had 3 to 7 transverse septa and 0 to 4 longitudinal septa. Beaks were broadly tapered. Sequence of the internal transcript spacer (ITS) region of isolate 22-094-A (GenBank Accession No. OP481973) had 100% homology to that of CBS 116495 (KC584190), a representative strain of A. cinerariae (Woudenberg et al. 2013). Based on the morphological and sequence characters, the casual fungus was identified as A. cinerariae. Pathogenicity confirmation was done in two separate assays. In a detached-leaf assay, mature leaves were collected from 5-year-old F. japonicum 'Gigantea' plants. Five leaves (abaxial surface) were sprayed with a mixture of conidial suspensions of the three isolates at 300 conidia per mL and 1.5 mL per leaf, while sterile water was used for a non-inoculated control leaf. Leaves were placed in a plastic tray with wet paper towels. The tray was placed at 22°C for an 8-h photoperiod and covered for 3 days to maintain moisture. Small purple to brown spots were visible on inoculated leaves 2 days after inoculation (DAI). More than 90% of inoculated leaf areas were blighted 10 DAI, whereas the control leaf remained asymptomatic. In a whole-plant assay, three F. japonicum 'Argenteo Marginata' plants grown in 10-inch pots were placed in a plastic tray and sprayed with a conidial suspension of 22-094-A onto both abaxial and adaxial surfaces at 300 conidia per mL and 40 mL per plant. The tray was maintained as described above. Sterile water was used for a non-inoculated control plant. Small leaf spots appeared on the inoculated plants 2 DAI. Large necrotic areas developed on leaves and girdled petioles causing aboveground tissues to collapse 4 DAI. All inoculated leaves were blighted 7 to 10 DAI, while the non-inoculated control plant remained healthy. Each assay was repeated once. Alternaria cinerariae, identified by distinct morphology traits (Nishikawa and Nakashima 2015), was consistently re-isolated from inoculated leaves in both assays. Leaf spot on F. japonicum caused by A. cinerariae has been reported in CA, USA (Woudenberg et al. 2013) and Japan (Sakoda et al. 2010). This is the first report in SC, USA. This fungus also infects at least 25 other hosts (Farr and Rossman 2022). This disease may pose a threat to leopard plants in nurseries and landscapes under conducive conditions. Disease management strategies are warranted.

Shared phytochemicals predict efficacy of essential oils against western flower thrips (Frankliniella occidentalis) in the greenhouse

Western flower thrips (Frankliniella occidentalis; Thysanoptera: Thripidae), or WFT, are a global pest of commercial crops, particularly those grown in greenhouses. Current management recommendations often involve judicious use of pesticides to which WFT have evolved multiple resistance phenotypes. Essential oils (EOs) have shown promise as a less toxic alternative for WFT greenhouse management. However, challenges remain in predicting which EOs are most likely to be insecticidal to WFT and ensuring that the efficacy of EOs under bioassay conditions reflect performance in whole-plant application scenarios. To address these challenges, 9 EOs were tested for contact toxicity against WFT in small container assays, then gas chromatography–mass spectroscopy (GC–MS) profiles of each EO were used to quantify concentrations of 22 chemicals shared by at least 5 or more of the plant species. Of these, 13 compounds were positively correlated with thrips mortality. Effective compounds were a mixture of sesquiterpenes, cyclic monoterpenes, and noncyclic monoterpenes. Interestingly, no bicyclic monoterpenes shared among the essential oils tested correlated with thrips mortality. Whole-plant assays of the four best EOs from the container assay showed significant reduction in the number of thrips per plant, although mortality in EO treatments in the whole plant assay was lower than in the container assay. In addition, all four EOs were as efficacious as the conventional insecticide flonicamid. Identifying other EOs with high concentrations of the efficacious compounds that were identified in this study and using container assays to screen these oils for WFT thrip mortality and phytotoxicity could help integrated pest management (IPM) practitioners and greenhouse staff to more rapidly accumulate a suite of EOs as low toxicity alternatives for management of WFT in greenhouse settings.Graphical

Up-regulation of bZIP88 transcription factor is involved in resistance to three different herbicides in both Echinochloa crus-galli and E. glabrescens.

The resistance of weeds to herbicides poses a major threat to agricultural production, and non-target-site resistance (NTSR) is often a serious problem as its mechanisms can in some cases confer resistance to herbicides with different modes of action. In this study, we hypothesized that bZIP transcription factors (TFs), which regulate abiotic stress responses in many plants, play a regulatory role in NTSR. Whole-plant assays indicated that the wild grasses Echinochloa crus-galli and E. glabrescens are resistant to the herbicides penoxsulam, cyhalofop-butyl, and quintrione. Transcriptome sequencing then identified 101 and 49 bZIP TFs with differential expression following penoxsulam treatment in E. crus-galli and E. glabrescens, respectively. Twelve of these genes had >60% homology with rice genes. The expression of bZIP88 was considerably up-regulated 6 h after treatment with the three different herbicides, and it was similar between resistant and susceptible populations; however, the relative expression levels before herbicide treatment and 24 h after were the same. We used rice (Oryza sativa ssp. japonica cv Nipponbare) as a model system for functional validation and found that CRISPR-Cas9-knockout of the rice bZIP88 ortholog increased the sensitivity to herbicide, whereas overexpression reduced it. The OsbZIP88 protein was localized to the nucleus. Using ChIP coupled with high-throughput sequencing, OsbZIP88 was found to form a network regulatory center with other TFs such as bZIP20/52/59 to regulate OsKS1, OsCOE1, and OsIM1, which are related to auxin, abscisic acid, brassinosteroids, and gibberellic acid. Based on these results, we have established a database of bZIP TFs corresponding to herbicide stress, and resolved the mechanisms of the positive regulation of herbicide resistance by bZIP88, thereby providing new insights for NTSR.

First Report of Phytophthora nicotianae Causing Dianthus chinensis Root Rot and Foliage Blight in China.

Dianthus chinensis is a popular ornamental plant that is widely cultivated in China. From May 2020 to 2021, root rot and foliage blight were observed on approximately 50% groundcover plants at several landscape sites of Xuanwuhu Park and Nanjing Railway Station, China. Symptoms of wilting and chlorosis appeared in the initial stage, and severe infection caused the whole plant to die . To recover the causal pathogen, infected root and leaf samples were cut into 5×5 mm2 squares, surface-disinfected in 70% ethanol for 30 sec, placed onto 10% clarified V8 PARP agar at 25°C . After three days, Phytophthora-like hyphae were visibly emerged from both root and leaf tissues and growing into cV8A. Individual hyphal tips were transferred to new cV8A plates to obtain a total of 10 pure isolates. Colony morphology of all isolates on cV8A had slightly radiate to stellate patterns with cottony aerial mycelia. After four or five days all isolates had identical morphological traits including papillate and noncaducous sporangia on cV8, hyphal swellings, and intercalary and terminal chlamydospores. A representative isolate Pni-dc7 was examined for morphological measurements. Sporangia were mostly ovoid and sometimes obpyriform, averaging 28.9±5.6 µm in length and 24.9±5.8 µm in width (n=30). Chlamydospores were abundant and spherical with an average diameter at 29.2 ± 0.3 µm (n=30). Oogonia were not observed. For sequence analysis, the internal transcribed spacer (ITS) regions and large subunit (LSU) of the nuclear ribosomal RNA gene complex were amplified using the primer pairs ITS1/ITS4 and NL1/NL4 , respectively, while the mitochondrial cytochrome c oxidase subunit II (coxII) gene was amplified using FM58/FM66 (Martin et al. 2003). The ITS sequence of isolate Pni-dc7 (GenBank Acc. No. MZ519893) had a 100% identity to those of P. nicotianae (MH219914, KU172524, MT065839). The LSU sequence (MZ573547) had a 100% identity to those of P. nicotianae (KX250514, MZ348950, HQ665198).The cox2 sequence (MZ519893) had a 100% identity to those of P. nicotianae (MH221078, KJ506439, JF707072). Based on morphological and molecular evidence, Pni-dc7 was identified as P. nicotianae. Pathogenicity tests were conducted using both detached leaves and whole plants. Asymptomatic leaves were collected from healthy plants.A 5×5 mm2 Pni-dc7-colonized cV8A plug was placed on each wound of five leaves. Sterile agar plugs were used for a non-inoculated control leaf. All six leaves were placed on a wet filter paper in a closed container at 25°C. All inoculated leaves had necrotic tissues around the wounds, the symptoms progressed from spots to the entire leaves after two days . The control leaves remained asymptomatic. In the whole-plant assay, a D. chinensis plant (approx. 0.3 m in height) was inoculated with 5 mL of zoospore suspension that was mixed into the potting soil(500g). Three plants were inoculated and control plants were treated with sterile distilled water. After two weeks all three inoculated plants in three repeats of the assay had root and crown rot and foliage blight, whereas all control plants remained asymptomatic. P. nicotianae was reisolated from all inoculated plants. This is the first report of P. nicotianae causing root rot and foliage blight on D. chinensis in China. Considering the importance of D. chinensis to both ornamental nursery and landscaping industries in China, diseased plants at the landscape sites were removed to prevent the spread of P. nicotianae to production sites and other landscape locations.

Comparison of Bioassays Used to Determine Onion Thrips (Thysanoptera: Thripidae) Susceptibility to Spinetoram.

Onion thrips (Thrips tabaci Lindeman) is one of onion's most damaging insect pests and has a history of developing resistance across insecticide classes. The susceptibility of T. tabaci populations to insecticides can be determined using laboratory bioassays. Three types of bioassays have been documented in the literature specifically for use with T. tabaci: vial assay (contact only), feeding assay (ingestion only), and leaf-dip assay (contact + ingestion). The objectives of this study were to 1) compare insecticide susceptibility levels of a T. tabaci population using these three assays and 2) determine which bioassay's results were most similar to those generated from exposing thrips to whole plants treated with insecticide. All experiments were conducted using a colony of T. tabaci known to be susceptible to insecticides and all were evaluated for their susceptibility to spinetoram (Radiant SC). Results indicated that 1) each bioassay generated a unique concentration-mortality relationship and LC50 value (0.01, 0.03 and 1.6 ppm for leaf-dip, vial, and feeding assays, respectively), and 2) all bioassays overestimated the susceptibility of the population relative to the whole-plant assay (LC50 = 5.3 ppm). Attributes of these bioassays are discussed relative to their future use in insecticide resistance monitoring programs for T. tabaci.

Location, location, location: Feeding site affects aphid performance by altering access and quality of nutrients.

Aphid feeding behavior and performance on a given host plant are influenced by the plants’ physical and chemical traits, including structural characters such as trichomes and nutritional composition. In this study, we determined the feeding behavior and performance of soybean aphids (Aphis glycines) on the stem, the adaxial (upper), and the abaxial (lower) leaf surfaces during early vegetative growth of soybean plants. Using the electrical penetration graph technique, we found that aphids feeding on the stem took the longest time to begin probing. Once aphids began probing, the sieve elements were more conducive to feeding, as evidenced by less salivation on the stem than either leaf surface. In whole-plant assays, stems harbored higher aphid populations, and aphids had shorter development time on stems than the adaxial and the abaxial leaf surfaces. We compared trichome density and length on the stem, the adaxial, and the abaxial leaf surfaces to investigate whether plant trichomes affected aphid feeding and performance. There were higher density and longer trichomes on stems, which likely resulted in aphids taking a longer time to probe. Still a negative impact on aphid population growth was not observed. Analysis of phloem sap composition revealed that vascular sap-enriched exudates from stems had higher sugars and amino acids than exudates from leaves. In artificial diet feeding assays, the population of aphids reared on a diet supplemented with stem exudates was higher than on a diet supplemented with leaf petiole exudates which is in agreement with results of the whole-plant assays. In summary, our findings suggest that the performance of soybean aphids on a specific plant location is primarily driven by accessibility and the quality of phloem composition rather than structural traits.

First Report of Phytophthora pini Causing Foliage Blight and Shoot Dieback of Rhododendron pulchrum in China.

During a 2019-2020 survey for plant pathogenic oomycetes in Nanjing, China, severe foliage blight and dieback were observed on approximately 20 Rhododendron pulchrum plants at three public parks and gardens. Approximately 25% of leaves and shoots were affected. Symptoms included brown to black lesions on leaves and stems, dieback of shoot tips, and wilting. Diseased tissues were collected from a five-year-old shrub with typical disease symptoms at Xuanwuhu Park. They were cut into 10×10 mm2 squares, immersed in 70% ethanol for 30 sec, and placed onto fresh clarified V8 juice agar (cV8A) containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene. Phytophthora-like hypae were transferred to new cV8A plates daily. A total of five isolates were obtained after five days of incubation at 25°C. After approximately 20 days, all isolates were identical in morphological traits including semi-papillate sporangia and gametangia (homothallic). Thirty sporangia of a representative isolate Ppi were randomly selected and examined. They were mostly ovoid and sometimes obpyriform, averaging 41.0 ± 3.9 × 24.8 ± 3.2 µm. Antheridia of 30 randomly selected gametangia were paragynous, averaging 16.7 ± 0.7 × 12.4 ± 1.5 µm. Average diameters of oogonia and plerotic oospores were 29.2 ± 0.3 µm and 26.4 ± 1.6 µm, respectively. Chlamydospores were not observed. The above morphological traits suggested the causal agent belonging to the "P. citricola-complex". Isolate Ppi was subjected to sequencing of the rDNA internal transcribed spacer (ITS) region and theras-related GTP-binding protein 1 (Ypt1) gene. ITS sequence of Ppi (GenBank ACN. MT672594) has 100% identity to that of P. pini (MG865565). It has a 3-nt difference from the ITS sequences of P. acerina (MG518642) and P. citricola (MG865475) and a 4-nt difference from that of P. plurivora (FJ665225). Ypt1 sequence of Ppi (MT680000) has 100% identity to that of P. pini (MK058416). Pathogenicity of Ppi on R. pulchrum was tested using both detached-leaf and whole-plant assays. In the former assay, each of six asymptomatic leaves was symmetrically wounded at both sides using a sterile inoculation needle. A 5×5 mm2 Ppi-colonized cV8A plug was placed on each wound of five leaves. Sterile agar plugs were used for a control leaf. All six leaves were placed on a wet filter paper in a closed container at 25°C. This assay was repeated twice. On the fifth day, all inoculated leaves had necrotic tissues around the wounds, while the control leaves remained asymptomatic. In the whole-plant assay, 20-inch-tall plants were used. Five attached leaves and the twig base of each plant were wounded. A control plant was inoculated in the same manner above, while sterile agar plugs were used. Each plant was covered with a plastic bag and maintained at 25°C. Wet cotton balls were placed in the bags to maintain humidity. After two days, the bag containing cotton balls was removed. This assay was repeated three times. After two weeks, all three inoculated plants in the three replicated trials had severe foliage blight and dieback, whereas control plants remained healthy. Phytophthora isolates recovered from artificially inoculated tissues were identical to isolate Ppi in morphological characters. Rhododendron diseases caused by P. pini were reported in the USA and Finland . This is the first report of P. pini causing foliage blight and dieback on R. pulchrum, an important nursery and landscape plant in China. Additional surveys are ongoing to determine the distribution of this pathogen in Nanjing. Management programs are under development to contain the spread of P. pini and treat diseased plants.

Plant hairy roots enable high throughput identification of antimicrobials against Candidatus Liberibacter spp.

A major bottleneck in identifying therapies to control citrus greening and other devastating plant diseases caused by fastidious pathogens is our inability to culture the pathogens in defined media or axenic cultures. As such, conventional approaches for antimicrobial evaluation (genetic or chemical) rely on time-consuming, low-throughput and inherently variable whole-plant assays. Here, we report that plant hairy roots support the growth of fastidious pathogens like Candidatus Liberibacter spp., the presumptive causal agents of citrus greening, potato zebra chip and tomato vein greening diseases. Importantly, we leverage the microbial hairy roots for rapid, reproducible efficacy screening of multiple therapies. We identify six antimicrobial peptides, two plant immune regulators and eight chemicals which inhibit Candidatus Liberibacter spp. in plant tissues. The antimicrobials, either singly or in combination, can be used as near- and long-term therapies to control citrus greening, potato zebra chip and tomato vein greening diseases.

Detached leaf inoculation assay for evaluating resistance to the spinach downy mildew pathogen

Downy mildew, caused by the obligate oomycete Peronospora effusa (Pfs), is the most economically important disease of spinach. In recent years, numerous new races of the pathogen have overcome the resistance used in newly released cultivars. Resistance to Pfs in spinach is predominantly governed by dominant major resistance genes (RPFs) that are widely used in commercial spinach hybrid cultivars. Currently, a labor and resource-intensive inoculation test of whole plants, in a large tray format, in a temperature-controlled growth chamber and dew chamber, is employed to evaluate the resistance of spinach germplasm and to characterize races of the downy mildew pathogen. The objective of this work was to evaluate, standardize, and validate a more efficient detached leaf inoculation method to differentiate resistant and susceptible spinach genotypes and characterize Pfs races on a standardized set of spinach differential genotypes. Detached leaves and cotyledons of standard host differentials commonly used for race differentiation were placed on water agar in closed Petri dishes and inoculated by spraying the leaves with a spore suspension of Pfs inoculum. Disease incidence and severity on detached leaves and cotyledons were compared to the response of the corresponding cultivars in the standard whole-plant assay. There was a complete match between the disease reaction on whole plants and the disease reaction on detached leaves for all three races of Pfs examined. Furthermore, the obligate pathogen could infect, sporulate, and maintain pathogenicity by propagation solely on detached leaves. The detached leaf assay could facilitate advances in breeding for Pfs by evaluating resistance, pathogen race identification, and studies on epidemiology and genetics of the pathogen as the tests are less labor, resource-intensive than the whole-plant assay format, and environmental variables can be more accurately controlled.

Cultivar variation for imazamox resistance in wheat (Triticum aestivum L.): Insights into enzymatic assays for early selection

Acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6) is the target site of several herbicide classes including imidazolinones. Imidazolinone resistance in wheat is conferred by two major genes AhasL-D1 and AhasL-B1. The objective of this work was to evaluate the in vitro and in vivo AHAS activity and plant growth in response to imazamox of nine wheat cultivars. Dose-response curves for two-gene resistant cultivars were significantly different from the single-gene resistant and susceptible cultivars in the in vitro AHAS assay. Resistance levels at the in vivo AHAS and whole-plant assays for resistant cultivars were >10-fold higher than susceptible cultivars. Moreover, in vivo dose-response curves showed differences among cultivars with the same number of resistance genes. It was concluded that in the in vitro AHAS assay cultivar variability was due to differences in target-site sensitivity while the in vivo AHAS assay reflected the resistance at whole-plant level. Both in vitro and in vivo AHAS dose-response curves could be useful tools when exploring mechanisms involved in imidazolinone resistance in different wheat genetic backgrounds and for the selection of higher resistant genotypes.

Indications of Susceptibility to Calonectria pseudonaviculata in Some Common Groundcovers and Boxwood Companion Plants

Knowing the host range of a pathogen is critical to developing and implementing effective disease management programs. Calonectria pseudonaviculata (Cps) is known to attack a number of species, varieties, and cultivars in the genus Buxus as well as three Pachysandra species (Pachysandra terminalis, Pachysandra procumbens, and Pachysandra axillaris) and several Sarcococca species, all in the Buxaceae family. The objective of this study was to evaluate non-Buxaceae groundcovers and companion plants commonly associated with boxwood plantings for their susceptibility to Cps. Twenty-seven plant species belonging to 21 families were exposed to different levels of inoculum: 50 to 300 conidia per drop for detached leaf assays and 30,000 to 120,000 conidia per 1 ml for whole-plant assays. Inoculated plants were incubated in humid environments for at least 48 h to facilitate infection. Cps infection and sporulation were observed on 12 plant species: Alchemilla mollis, Arctostaphylos uva-ursi, Brunnera macrophylla, Epimedium × youngianum, Galium odoratum, Geranium sanguineum, Phlox subulata, Tiarella cordifolia, Callirhoe involucrata, Iberis sempervirens, Mazus reptans, and Vinca minor. These results suggest that there may be more hosts of Cps commonly grown in nurseries and landscapes. If corroborated by observations of natural infection, these findings have implications for the Boxwood Blight Cleanliness Program instituted by the National Plant Board and for planning disease mitigation at production and in the landscape.

First evidence of multiple resistance of Sumatran Fleabane (Conyza sumatrensis (Retz.) E.Walker) to five- mode-of-action herbicides

Herbicide resistance is the evolutionary response of weeds to the selection pressure caused by repeated application of the same active ingredient. It can result from two different mechanisms, known as target site resistance (TSR) and non-target site resistance (NTSR). In addition to single-herbicide resistance, multiple resistance can occur due to herbicides selection or accumulation of resistance genes by cross-pollination. The aim of this research was to investigate the suspect of multiple herbicide resistance of Sumatran Fleabane (Conyza sumatrensis (Retz.) E.Walker) to herbicides frequently used as a burndown application. Dose-responses in a whole-plant assay were carried out to investigate multiple-resistance of Sumatran fleabane to paraquat, saflufenacil, diuron, 2,4-D and glyphosate. Results indicated that the resistance index (ratio R/S) based on herbicide rate to cause 50% mortality (LD50) were 25.51, 1.39, 7.29, 1.84 and 7.55 for paraquat, saflufenacil, diuron, 2,4-D and glyphosate, respectively. Based on herbicide rate required to cause a 50% reduction in plant growth (GR50), the resistant index were 51.83, 14.10, 5.05, 3.96 and 32.90 for the same herbicides, respectively. Our results confirmed multiple resistance of Conyza sumatrensis from Paraná-Brazil to herbicides from five-mode of-action. This was the first report of Conyza sumatrensis resistant to 2,4-D and the first case of Conyza sumatrensis presenting multiple resistant to herbicides from five- mode of-action in the world.

Extreme resistance to late blight disease by transferring 3 R genes from wild relatives into African farmer-preferred potato varieties

Three late blight resistance (R) genes from wild potato species were transferred as a stack into the farmer-preferred varieties ‘Tigoni’ and ‘Shangi’. Transgenic events were tested by detached-leaf assays (DLA) and whole-plant assays (WPA) with isolates of Phytophthora infestans using 20,000 sporangia / mL inoculum. For the first DLA, 9 out of 13 ‘Tigoni’ and 10 out of 12 ‘Shangi’ transgenic events had scores below 3% of leaf affected area. 17 of the 19 transgenic events with low scores were subjected to a second DLA using two different P. infestans isolates. 8 of them exhibited extreme LB resistance while the remaining 9 transgenic events showed hypersensitive response (HR). 6 transgenic events (2 ‘Tigoni’ and 4 ‘Shangi’) with extreme resistance by DLA were subjected to WPA and they all exhibited extreme resistance to LB. Hence, this study demonstrates that a simple DLA predicts high level of resistance to late blight. R gene expression analysis in 18 transgenic events showed different transgenic events exhibiting different levels of expression in the three genes. However, that pathogen induction and / or high R gene expression are necessary for extreme resistance when transgenic events bear a stack of three R genes was not observed. Key words: Detached-leaf assay, late blight, Phytophthora infestans, resistance.

Techniques for screening Cercospora leaf spot resistant fenugreek genotypes

Cercospora leaf spot (CLS) caused by Cercospora traversiana is an important phyto-pathological problem of fenugreek (Trigonella foenum-graecum L.), a multiuse legume crop. Field screenings for resistant plants, although accurate and effective, demand significant time and a sizable workforce to accomplish. Also, weather conditions in the field may not always be favourable for uniform disease spread, which eventually may lead to failure of the overall experiment. Whole-plant assays (WPA) and detached leaf assays (DLA) with artificial inoculation not only help in scaling up the number of plants screened but also reduce the space, time, and amount of inoculum needed for the experiment. The results from our two experiments indicate that both the WPA and DLA methods can be used reliably to differentiate resistant and susceptible genotypes of fenugreek. In addition, the correlation coefficient between WPA and DLA (r = 0.875, P < 0.01), derived from the mean disease score of each genotype, shows that they can be used interchangeably while screening fenugreek for CLS. DLA was found to be temperature-sensitive for the development of CLS symptoms and wounded leaves developed symptoms faster than non-wounded leaves. These indoor methods can be used for the development of CLS-resistant fenugreek cultivars in areas where disease development is difficult under field conditions.

A Bacterial Endophyte from Apoplast Fluids Protects Canola Plants from Different Phytopathogens via Antibiosis and Induction of Host Resistance.

Endophytic bacteria colonize inner plant tissues and thrive at the apoplast, which is considered its main reservoir. Because this niche is the place where the main molecular events take place between beneficial and pathogenic microorganisms, the aim of this work was to characterize culturable endophytic bacteria from apoplastic fluids obtained from field-grown canola leaves and analyze their potential for biological control of diseases caused by Xanthomonas campestris, Sclerotinia sclerotiorum, and Leptosphaeria maculans. Dual-culture analysis indicated that three isolates (Apo8, Apo11, and Apo12) were able to inhibit the growth of all three phytopathogens. Sequencing of the 16S ribosomal RNA and rpoD genes of these isolates revealed that they are closely related to Pseudomonas viridiflava. One of the isolates, Apo11, was able to diminish the propagation of X. campestris in whole-plant assays. At the same time, Apo11 inoculation reduced the necrotic lesions provoked by S. sclerotiorum on canola leaves. This protective effect might be due to the induction of resistance in the host mediated by salicylic and jasmonic acid signaling pathways or the production of compounds with antimicrobial activity. At the same time, Apo11 inoculation promoted canola plant growth. Thus, the isolate characterized in this work has several desirable characteristics, which make it a potential candidate for the formulation of biotechnological products to control plant diseases or promote plant growth.

Cross-Resistance of Eclipta (Eclipta prostrata) in China to ALS Inhibitors Due to a Pro-197-Ser Point Mutation

Eclipta, widespread in tropical, subtropical, and temperate regions, is one of the main malignant broadleaf weeds and thrives in moist and dryland fields. Field rates of acetolactate synthase (ALS) inhibitors have failed to control eclipta in some farmlands in China. One ALS inhibitor–resistant population (R) collected from Jiangsu province in China was confirmed in the greenhouse in our preliminary work. Whole-plant assays revealed that this R population was highly resistant to four sulfonylureas (pyrazosulfuron-ethyl, 134-fold; bensulfuron-methyl, 172-fold; metsulfuron-methyl, 30-fold; and tribenuron-methyl, 195-fold), two triazolopyrimidines (pyroxsulam, 98-fold; penoxsulam, 30-fold), and one pyrimidinylthio-benzoate (bispyribac-sodium, 166-fold) and was moderately resistant to two imidazolinones (imazethapyr, 10-fold; imazapic, 19-fold). ALS enzyme-activity assays showed insensitivity of the ALS from the R population (resistance index values ranged from 12 to 293) to all of the above ALS inhibitors in vitro. Chromatograms fromALSgene sequence analysis detected a homozygous Pro-197-Ser amino acid substitution in the R population. These results confirmed that the Pro-197-Ser substitution results in broad-spectrum cross-resistance to ALS inhibitors in the eclipta R population. To our knowledge, this study is the first to report broad cross-resistance to ALS inhibitors in eclipta and to obtain the full-lengthALSgene sequence.

Antifungal activity of sterols and dipsacus saponins isolated from Dipsacus asper roots against phytopathogenic fungi

The in vivo antifungal activity of crude extracts of Dipsacus asper roots was evaluated against the phytopathogenic fungi Botrytis cinerea, Colletotrichum coccodes, Blumeria graminis f. sp. hordei, Magnaporthe grisea, Phytophthora infestans, Puccinia recondita and Rhizoctonia solani using a whole-plant assay method. Ethyl acetate and acetone extracts, at 1000μg/mL, suppressed the development of tomato gray mold (TGM) and tomato late blight (TLB) by 90%. Through bioassay-guided isolation, five antifungal substances were isolated from the D. asper roots and identified as β-sitosterol (1), campesterol (2), stigmasterol (3), cauloside A (4) and a novel dipsacus saponin, named colchiside (3-O-β-d-xylopyranosyl-23-O-β-d-glucopyranosyl-28-O-β-d-(6-O-acetyl)-glucopyranosyl hederagenin) (5). Of those, cauloside A (4) displayed the greatest antifungal efficacy against rice blast, TGM and TLB. Colchiside (5) moderately suppressed the development of TLB, but exhibited little effect against the other diseases. The synergistic effects of the isolated compounds against TLB were also assessed. Synergistic and additive interactions were observed between several of the sterol compounds. This study indicated that the crude extracts of, and bioactive substances from, the roots of D. asper suppress TGM and TLB. In addition, cauloside A (4) and colchiside (5) could be used as antifungal lead compounds.