Spray Inoculation Research Articles

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

Screening for Susceptibility to Anthracnose Stem Lesions in Southern Highbush Blueberry

‘Flicker’ is a southern highbush blueberry (SHB, Vaccinium corymbosum) cultivar frequently selected by growers in Central and South Florida. In 2014, several growers in Central Florida experienced issues with anthracnose stem lesions and twig dieback on ‘Flicker’, resulting in a reduction in new plantings and the removal of many existing plantings. The objective of this study was to determine the level of anthracnose susceptibility of certain commercially available SHB cultivars, which information can be used to limit further use of susceptible cultivars in the University of Florida blueberry breeding program. The screening was performed using a spray inoculation of a virulent Colletotrichum gloeosporioides isolate onto whole V. corymbosum plants, followed by measurement of incidence and severity of disease over time. In repeated experiments, ‘Flicker’ and two other cultivars had a significantly higher mean number of lesions and area under the disease progress curve (AUDPC) than any other tested cultivar, and in both experiments, the observed lesions were similar in many respects to those previously reported on northern highbush blueberry (also V. corymbosum). Although the results of these experiments may ultimately indicate that Flicker has a unique genetic susceptibility to this form of anthracnose among SHB cultivars commercially grown in Florida, screening of additional cultivars must be performed for confirmation.

Genetic Analysis of Fusarium Head Blight Resistance in CIMMYT Bread Wheat Line C615 Using Traditional and Conditional QTL Mapping.

Fusarium head blight (FHB) is a destructive wheat disease present throughout the world, and host resistance is an effective and economical strategy used to control FHB. Lack of adequate resistance resource is still a main bottleneck for FHB genetics and wheat breeding research. The synthetic-derived bread wheat line C615, which does not carry the Fhb1 gene, is a promising source of FHB resistance for breeding. A population of 198 recombinant inbred lines (RILs) produced by crossing C615 with the susceptible cultivar Yangmai 13 was evaluated for FHB response using point and spray inoculations. As the disease phenotype is frequently complicated by other agronomic traits, we used both traditional and multivariate conditional QTL mapping approaches to investigate the genetic relationships (at the individual QTL level) between FHB resistance and plant height (PH), spike compactness (SC), and days to flowering (FD). A linkage map was constructed from 3,901 polymorphic SNP markers, which covered 2,549.2 cM. Traditional and conditional QTL mapping analyses found 13 and 22 QTL for FHB, respectively; 10 were identified by both methods. Among these 10, three QTL from C615 were detected in multiple years; these QTL were located on chromosomes 2AL, 2DS, and 2DL. Conditional QTL mapping analysis indicated that, at the QTL level, SC strongly influenced FHB in point inoculation; whereas PH and SC contributed more to FHB than did FD in spray inoculation. The three stable QTL (QFhbs-jaas.2AL, QFhbp-jaas.2DS, and QFhbp-jaas.2DL) for FHB were partly affected by or were independent of the three agronomic traits. The QTL detected in this study improve our understanding of the genetic relationships between FHB response and related traits at the QTL level and provide useful information for marker-assisted selection for the improvement of FHB resistance in breeding.

Can Additional Inoculations Increase Soybean Nodulation and Grain Yield?

Core Ideas A full set of N2–fixing nodules can supply soybean N demands.Seed inoculation increased nodulation and grain yield of field‐grown soybean.Additional spray inoculations (V1, V3, V6, R1, or R3) resulted in further increases.New nodules and grain yield increases can be achieved by additional inoculations. Highly productive soybean [Glycine max (L.) Merr.] genotypes require that large amounts of N be supplied throughout the growth cycle to attain high yields. The nutrient can be obtained through biological N2 fixation by elite bradyrhizobial strains if a set of fully functional nodules is available. This study was conducted during two crop seasons in the central‐west region of Brazil to investigate if additional inoculations via spray applications of bradyrhizobia at different stages of the growth cycle can result in increased nodulation and grain yield. Even though the soils had high populations of soybean bradyrhizobia, seed inoculation at sowing increased grain yield by 28 and 27% in the first and second seasons, respectively, compared to the non‐inoculated control. Additional inoculations performed until the R6 stage significantly increased soybean nodulation, which might indicate that plants were able to overcome the limitations of autoregulation of nodulation, allowing the development of new functional nodules to sustain the increased demand for N at latter stages of the growth cycle, especially during pod filling. In response to the increased nodulation, grain yield gains of 2 and 7% were obtained in the first and second experiments, respectively, relative to inoculation at sowing only. Our results indicate that soybean roots can form new nodules in response to additional inoculations, promoting increased N2 fixation and higher grain yield.

Development of screening technique for artificial creation of false smut in rice

False smut (Ustilaginoidea virens (Cooke) Takahashi) of rice is an emerging disease and a potential threat to rice growers in different countries due to reduction in yield, quality and production of mycotoxins. Development and cultivation of resistant varieties is desirable approach for its management. Different inoculation techniques viz. spray inoculation, syringe inoculation and dusting method were evaluated for creating artificial epiphytotic conditions in susceptible variety PR116 under natural as well as artificially provided humidity conditions. Spray inoculation method produced more number of smut balls per plant (3.21) and per cent infected panicles (33.44%) followed by syringe inoculation (2.00 and 26.57%, respectively) and dusting method (0.33 and 6.67%, respectively). However, syringe inoculation method produced more spikelet sterility (14.82%) in rice panicles. More disease occurrence was reported in plants provided with artificial humidity after inoculations (51.12% infected panicles) than the plants grown under natural conditions (13.33% infected panicles). Late transplanted crop developed higher disease severity (18.17% infected panicles and 3.34 smut balls/plant) which might be attributed by slight decrease in temperature and increase in relative humidity conditions (up to 88% RH) at time of infection from boot to early flowering stage of the rice crop.

Infection by Magnaporthe oryzae chrysovirus 1 strain A triggers reduced virulence and pathogenic race conversion of its host fungus, Magnaporthe oryzae

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is associated with an impaired growth phenotype of its host fungus, Magnaporthe oryzae. In this report, we assayed the virulence and pathogenicity of MoCV1-A-infected and MoCV1-A-free M. oryzae on rice plants. MoCV1-A infection did not affect virulence-associated fungal traits, such as conidial germination and appressorium formation. However, after punch inoculation of leaves on rice plants, MoCV1-A-infected strain formed smaller lesions than the MoCV1-A-free strain did on all rice varieties tested, showing that MoCV1-A infection resulted in reduced virulence of host fungi in rice plants. In contrast, after spray inoculation of rice seedlings, in some cases, MoCV1-A-infected and MoCV1-A-free strains caused different lesion types (resistance to susceptible, or vice versa) on individual international differential rice varieties. However, we did not find any gain/loss of the fungal avirulence genes by PCR, suggesting that MoCV1-A infection can convert the pathogenicity of the host M. oryzae from avirulence to virulence, or from virulence to avirulence, depending on the rice variety. We also confirmed the correlation of these race conversion events and invasive hyphae growth of the fungi in a leaf sheath inoculation assay. These data suggested that MoCV1-A infection generally confers hypovirulence to the fungal host and could be a driving force to generate physiological diversity, including pathogenic races.

Application of infrared thermal imaging for the rapid diagnosis of crop disease

Visual diagnosis of plant disease following a visually evident epidemic results in untimely and excessive application of pesticide. Conventional nondestructive testing methods are difficult for early recognition before the onset of symptoms. In this study, tomato mosaic disease and wheat leaf rust were taken as examples. Infrared thermal imaging technology was used to continuously detect the temperature information of the two crops during the incubation period following the introduction of inoculum. Diseases of tomato and wheat were induced by rubbing inoculation and spray inoculation, respectively. Maximum temperature difference (MTD) was calculated to characterize the continuous temperature change during the incubation period. The results showed that the MTD of tomato mosaic disease ranged from 0.2°C ~ 1.7°C, and that of wheat leaf rust 0.4°C ~ 2°C. As the disease progresses, the MTD of both plants showed an increasing trend while the average temperature decreased. The time to observe the symptoms earlier than the naked eye for tomato mosaic and wheat leaf rust is 5d and 7d respectively. Studies showed that it is feasible to realize the early determination of disease through the combination of infrared thermal imaging and MTD calculation. It provides the theoretical basis and method basis for the early and accurate application of spraying.

Arbuscular mycorrhiza‐mediated resistance in tomato against Cladosporium fulvum‐induced mould disease

AbstractRoot colonization with arbuscular mycorrhizal fungi (AMF) enhances plant resistance particularly against soil‐borne pathogenic fungi. In this study, mycorrhizal inoculation with Glomus mosseae (Gm) significantly alleviated tomato mould disease caused by the air‐borne fungal pathogen, Cladosporium fulvum (Cf). The disease index (DI) in local leaves (receiving pathogen inoculation) and systemic leaves (just above the local leaf without pathogen inoculation) was 36.4% and 11.7% in mycorrhizal plants, respectively, whereas DI was 59.6% and 36.4% in the corresponding leaves of AMF non‐inoculated plants, after 50 days of Gm inoculation, corresponding to 15 days after Cf inoculation by leaf infiltration. Foliar spray inoculation with Cf also revealed that AMF pre‐inoculated plants had a higher resistance against subsequent pathogen infection, where the DI was 41.3% in mycorrhizal plants vs. 64.4% in AMF non‐inoculated plants. AMF‐inoculated plants showed significantly higher fresh and dry weight than non‐inoculated plants under both control (without pathogen) and pathogen treatments. AMF‐inoculated plants exhibited significant increases in activities of superoxide dismutase and peroxidase, along with decreases in levels of H2O2 and malondialdehyde, compared with non‐inoculated plants after pathogen inoculation. AMF inoculation led to increases in total chlorophyll contents and net photosynthesis rate as compared with non‐inoculated plants under control and pathogen infection. Pathogen infection on AMF non‐inoculated plants led to decreases in chlorophyll fluorescence parameters. However, pathogen infection did not affect these parameters in mycorrhizal plants. Taken together, these results indicate that AMF colonization may play an important role in plant resistance against air‐borne pathogen infection by maintaining redox poise and photosynthetic activity.

Phenotypic and biochemical characterization of new advanced durum wheat breeding lines from Algeria that show resistance to fusarium head blight and to mycotoxin accumulation

Durum wheat (Triticum turgidum L. var. durum) is a crop highly susceptible to Fusarium head blight (FHB), which results in yield losses and downgrades the quality of grains mostly due to mycotoxin contamination. Although breeding for resistance to FHB is one of the most promising strategy for minimizing crop damage, the attempts to obtain durum wheat resistant lines have been limited so far. Two potentially interesting lines were recently delivered by a breeding program based on crosses involving cultivars from Europe and Syria. Using a field experiment in Algeria with four different Fusarium culmorum strains and a spray inoculation method, we demonstrated that the two breeding lines were significantly less affected than a set of commercial cultivars. The two breeding lines were shown to exhibit a higher resistance to both initial fungal infection and disease spread , and to mycotoxin contamination. In addition, a detailed analysis of 2 phenolic acid composition of grains indicates significant differences in p-coumaric acid between the FHB-resistant breeding lines and the sensitive genotypes. Our results corroborate the importance of cell wall composition in preventing the diffusion of F. culmorum and therefore contributing to the resistance of cereals to FHB.

Transgenic expression of antimicrobial peptide D2A21 confers resistance to diseases incited by Pseudomonas syringae pv. tabaci and Xanthomonas citri, but not Candidatus Liberibacter asiaticus.

Citrus Huanglongbing (HLB) associated with ‘Candidatus Liberibacter asiaticus’ (Las) and citrus canker disease incited by Xanthomonas citri are the most devastating citrus diseases worldwide. To control citrus HLB and canker disease, we previously screened over forty antimicrobial peptides (AMPs) in vitro for their potential application in genetic engineering. D2A21 was one of the most active AMPs against X. citri, Agrobacterium tumefaciens and Sinorhizobium meliloti with low hemolysis activity. Therefore, we conducted this work to assess transgenic expression of D2A21 peptide to achieve citrus resistant to canker and HLB. We generated a construct expressing D2A21 and initially transformed tobacco as a model plant. Transgenic tobacco expressing D2A21 was obtained by Agrobacterium-mediated transformation. Successful transformation and D2A21 expression was confirmed by molecular analysis. We evaluated disease development incited by Pseudomonas syringae pv. tabaci in transgenic tobacco. Transgenic tobacco plants expressing D2A21 showed remarkable disease resistance compared to control plants. Therefore, we performed citrus transformations with the same construct and obtained transgenic Carrizo citrange. Gene integration and gene expression in transgenic plants were determined by PCR and RT-qPCR. Transgenic Carrizo expressing D2A21 showed significant canker resistance while the control plants showed clear canker symptoms following both leaf infiltration and spray inoculation with X. citri 3213. Transgenic Carrizo plants were challenged for HLB evaluation by grafting with Las infected rough lemon buds. Las titer was determined by qPCR in the leaves and roots of transgenic and control plants. However, our results showed that transgenic plants expressing D2A21 did not significantly reduce Las titer compared to control plants. We demonstrated that transgenic expression of D2A21 conferred resistance to diseases incited by P. syringae pv. tabaci and X. citri but not Las. Our results underscore the difficulty in controlling HLB compared to other bacterial diseases.

Inoculation treatments affect the migration and colonisation of rhizobia in alfalfa (Medicago sativa L.) plants

ABSTRACTPurpose: The purposes of this study were to characterise the migration and the colonisation dynamics of two different fluorescent-tagged rhizobia in various alfalfa tissues (especially in seeds); and also to develop efficient inoculation treatments to promote colonisation ability of target rhizobia in elite seed varieties.Materials and methods: Four treatments (root drench, root damaging and drench, root drench with matrine, and flower spray) were applied to inoculate alfalfa with two fluorescent-tagged rhizobia, Ensifer meliloti LZgn5f (gn5f) and Ensifer meliloti 12531f (12531f), at three different growth stages; bud, flower and pod stages. The migration and colonisation dynamics of the two fluorescent tagged rhizobia strains were monitored using UV lamp detection and a stereo fluorescence microscopy.Results: The results showed that both rhizobia strains mainly colonised the roots and could migrate to aerial tissues. In aerial tissues, when alfalfa plants were inoculated during the bud stage, both rhizobia strains mainly colonised the leaves and stems; during the flower stage, a spray inoculation treatment resulted in more 12531f colonising reproductive tissues, while during the pod stage, more rhizobial strains gn5f colonised seeds using the root drench with matrine treatment.Conclusions: These results indicate that endophytic rhizobia are natural inhabitants of internal regions of roots, stems, leaves and that the endophytes may arise from reproductive tissues, such as seeds. Understanding the population dynamics of endophytic rhizobia in alfalfa would considerably improve the survival of target rhizobia during seed transfer. Combining target endogenous rhizobial species with good alfalfa seed varieties may lead to the development of a novel breeding method.

Further Evidence of Cucurbit Host Specificity among Acidovorax citrulli Groups Based on a Detached Melon Fruit Pathogenicity Assay.

Bacterial fruit blotch, caused by the gram-negative bacterium Acidovorax citrulli, is a serious economic threat to cucurbit crop production worldwide. A. citrulli strains can be divided into two genetically distinct groups, with group I strains infecting a range of cucurbit species and group II strains being predominantly associated with watermelon. Group I and II A. citrulli strains differ in their arsenal of type III secreted (T3S) effector proteins and we hypothesize that these effectors are critical for cucurbit host preference. However, the pathogenicity or virulence assays used for A. citrulli, including infiltration of seedling cotyledons and mature fruit rind tissues with cell suspensions and spray inoculation of seedlings, lack the sensitivity to consistently distinguish strains of the two groups. Here, we describe an immature, detached melon fruit assay based on 'Joaquin Gold' melon (Syngenta, Rogers Brand) that clearly indicates differences in host specificity between group I and II A. citrulli strains. Using this assay, four group I strains (M6, AAC213-52, AAC213-55, and XJL12) induced typical water-soaked lesions in melon fruit rind tissue 7 to 10 days after pinprick inoculation. In contrast, four group II strains (AAC00-1, AAC213-44, AAC213-47, and AAC213-48) did not induce water-soaked lesions on detached melon fruit rinds during the same period. These data suggest that group I A. citrulli strains have a specific capacity to infect immature Joaquin Gold melon fruit, whereas group II strains do not. Interestingly, this differential pathogenicity phenotype was not observed on foliar seedling tissues of the same melon cultivar, suggesting that host preference of A. citrulli strains is specific to immature fruit tissues. Using the immature melon fruit inoculation assay, a T3S system mutant of the group I A. citrulli strain, M6 (M6ΔhrcV), failed to induce water soaking. This indicates that T3S effectors are involved in A. citrulli cucurbit host preference, and that this assay is suitable for future studies of unique T3S effectors that distinguish group I and II strains.

Paraphoma chlamydocopiosa sp. nov. and Paraphoma pye sp. nov., two new species associated with leaf and crown infection of pyrethrum

Two new pathogens of pyrethrum, described as Paraphoma chlamydocopiosa and Paraphoma pye, isolated from necrotic leaf lesions on pyrethrum plants in northern Tasmania, Australia, were identified using morphological characters, phylogenetic analysis of the internal transcribed spacer (ITS), elongation factor 1‐α (EF1‐α) and β‐tubulin (TUB) genes, and pathogenicity bioassays. Bootstrap support in the combined and individual gene region phylogenetic trees supported the two species that were significantly different from the closely related P. chrysanthemicola and P. vinacea. Morphological characteristics also supported the two new species, with conidia of P. chlamydocopiosa being considerably longer and wider than either P. chrysanthemicola or P. vinacea, and P. pye being distinct in forming bilocular pycnidia. Glasshouse pathogenicity tests based on root dip inoculation resulted in P. chlamydocopiosa and P. pye infecting the crown and upper root tissues of pyrethrum plants, and significant reduction in biomass 2 months after inoculation. Both of these Paraphoma species caused leaf lesions during in vitro and in vivo bioassays 2 weeks after foliar spray inoculation. Although P. chlamydocopiosa and P. pye were shown to be crown rot pathogens, they were also commonly isolated from leaves of diseased plants in pyrethrum fields of northern Tasmania.

Enhancing Resistance to Fusarium graminearum in Danish and Iraqi Local Winter Wheat Varieties under Greenhouse Conditions

Fusarium head blight (FHB), caused by Fusarium graminearum, has become one of the most destructive diseases in the world’s wheat growing areas, especially in the humid and semi humid regions. F. graminearum results in yield and quality reduction and accumulation of mycotoxins. Nine winter wheat cultivars widely grown in Denmark or Iraq have been evaluated for FHB resistance during 2014/2015. The evaluation was made by means of artificial inoculations with F. graminearum under greenhouse conditions. Two inoculation methods were used Injection method, detect type II resistance by injecting fusarium conidia into individual florets and detect resistance to initial infection type I resistance by Spraying Fusarium conidia on the spikes. More precise data relating the effects of FHB on yield have been obtained using inoculated trials. Based on these data, there was no significant difference between syringe inoculation and spray inoculation (p<0.0001), whereas a significant differences between cultivars and method of inoculation on cultivars, Eiba99 and Rabiea which were assessed as resistant, and the cultivars, Skalmeje, Ritmo, Sham6, Aras, Mariboss, Eiba95 and Hereford as susceptible to FHB. Response to FHB was assessed by the percentage of visually infected spikelets and head weight relative to the non-inoculated (control). In the resistant cultivars, both the total grain yield and some of the yield elements (spike weight, grain /spike and 1000 kernel weight) were significantly less affected by FHB than in susceptible cultivars. Furthermore, the yield components (spike weight, grain number /spike, grain weight/spike, TKW) were significantly and negatively affected by artificial inoculation with FHB in all nine tested wheat cultivars. On the basis of experimental results it is concluded that artificial inoculation with F. graminearum could provide additional information about selection of both resistant and susceptible wheat cultivars.

Comparison of Spray and Point Inoculation with &amp;lt;i&amp;gt;Fusarium graminearum&amp;lt;/i&amp;gt; to Evaluate FHB Disease in Two Winter Wheat Genotypes Under Temperature Stress

Two near isogenic lines of wheat; Mercia 1 (Rht-B1b) and Mercia 2 (Rht-D1b) were evaluated for Type I and Type II resistance using point or spray inoculation and incubated under controlled environment. The experiment was a complete factorial combination of 2 x 4 x 2 x 4 {2 genotypes, 4 inoculation treatments (spray + <i>Fusarium</i>, spray + SDW, point + <i>Fusarium</i>, point + SDW) x 2 temperatures} and 4 randomised replicates. Wheat spikes were sprayed with a single spore isolate of <i>F. graminearum</i> 4 days after the start of flowering and transferred to controlled environment cabinets set at either 23/15°C or 28/20°C for 14 days and then taken outside to mature. Results reveals that genotype showed no difference in FHB severity in both spray inoculation and point inoculation but the temperature main effect only approached significance (P=0.071) with low temperature increasing FHB severity following point inoculation. High temperature significantly (P&lt;0.001) increased DON concentration in spray inoculation, contrasted the effect in point inoculation. However, the amount of DON per grain showed no significant (P&gt;0.05) effect. Grains per spike was significantly (P&lt;0.011) reduced by 25% following spray when compared with point inoculation, but showed no significant (P&gt;0.05) effect of temperature. On average, <i>Fusarium</i> infection significantly (P&lt;0.001) reduced wheat grain weight by 28% when compared with uninoculated control.

Race Typing of Puccinia striiformis on Wheat.

A procedure for virulence phenotyping of isolates of yellow (stripe) rust using spray inoculation of wheat seedlings by spores suspended in an engineered fluid, Novec™ 7100, is presented. Differential sets consisting of near-isogenic Avocet lines, selected lines from the "World" and "European" sets, and additional varieties showing race-specificity facilitate a robust assessment of race, irrespectively of geographical and evolutionary origin of isolates. A simple procedure for purification of samples consisting of multiple races is also presented.

Further Characterization of Genetically Distinct Groups of Acidovorax citrulli Strains.

Bacterial fruit blotch of cucurbits (BFB) is caused by the gram-negative bacterium Acidovorax citrulli, whose populations can be distinguished into two genetically distinct groups, I and II. Based on visual assessment of BFB severity on cucurbit seedlings and fruit after inoculation under greenhouse conditions, group I A. citrulli strains have been reported to be moderately to highly virulent on several cucurbit hosts, whereas group II strains have exhibited high virulence on watermelon but low virulence on other cucurbits. Additionally, group I strains are recovered from a range of cucurbit hosts, while group II strains are predominantly found on watermelon. The goal of this research was to develop tools to characterize and rapidly distinguish group I and II A. citrulli strains. We first sought to determine whether quantification of A. citrulli colonization of cucurbit seedling tissue reflects the differences between group I and II strains established by visual assessment of BFB symptom severity. Spray inoculation of melon seedlings with cell suspensions containing approximately 1 × 104 CFU/ml resulted in significantly higher (P = 0.01) population growth of M6 (group I; mean area under population growth curve [AUPGC] = 43.73) than that of AAC00-1 (group II; mean AUPGC = 39.33) by 10 days after inoculation. We also investigated the natural spread of bacterial cells and the resulting BFB incidence on watermelon and melon seedlings exposed to three group I and three group II A. citrulli strains under mist chamber conditions. After 5 days of exposure, the mean BFB incidence on melon seedlings exposed to representative group II A. citrulli strains was significantly lower (25 and 3.98% in experiments 1 and 2, respectively) than on melon seedlings exposed to representative group I strains (94.44 and 76.11% in experiments 1 and 2, respectively), and on watermelon seedlings exposed to representative group I and II strains (70 to 93.33%). Finally, we developed a polymerase chain reaction assay based on the putative type III secretion effector gene, Aave_2166, to rapidly distinguish group I and II A. citrulli strains. This assay will be important for future epidemiological studies on BFB.

Occurrence and identification of Stemphylium lycopersici causing Stemphylium leaf spot disease on tomato in Taiwan

Tomato leaves showing severe leaf spot symptoms have been observed and sampled in the central west and southwest Taiwan during 2015 and 2016. The symptoms were similar to those of bacterial leaf spot/late blight diseases, but only Stemphylium-like fungi were consistently isolated from the diseased tomato. Upon spray inoculation of tomato, Stemphylium-like isolates caused leaf spot symptoms identical to those of naturally infected plants, and the pathogenic isolates were successfully re-isolated from inoculated leaves. The tomato-pathogenic isolates were identified as S. lycopersici based on morphological characterization and molecular identification. S. lycopersici has been previously reported to cause gray leaf spot of tomato in the temperate regions, but the majority of S. lycopersici-caused lesions were black/dark brown rather than gray in our surveillance. Accordingly, it is suggested that S. lycopersici-caused disease of tomato is named Stemphylium leaf spot of tomato more appropriately than tomato gray leaf spot. Moreover, S. lycopersici-caused leaf spot disease on tomato has been distributed in major tomato production regions in Taiwan. The information provided by our study will be important for future breeding of tomato cultivars, especially for tomato producers in Taiwan.

Impact of primary infection site of Fusarium species on head blight development in wheat ears evaluated by IR-thermography

The effect of the primary infection site by Fusarium graminearum and F. culmorum within wheat ears on Fusarium head blight (FHB) was investigated under controlled conditions. FHB development was assessed visually and thermographically following inoculation by: (i) spraying ears, or injecting inoculum into spikelets on (ii) tip, (iii) centre and (iv) base of the ears, separately. Fusarium infection significantly increased the temperature span within ears 6 days post inoculation (dpi), especially infections starting at the ear tip. The temperature difference between air and ear was negatively correlated to FHB severity and enabled disease detection even 29 dpi. F. culmorum caused significant higher disease severity neither reflected in the frequency of infected kernels nor in thousand kernel weight (TKW). Spray inoculations had the strongest effect on TKW, whereas tip inoculations had no effect. Centre and base inoculations had intermediate effects on TKW, although FHB levels did not differ with the same trend among inoculation scenarios. The overall low correlations among FHB severity, infected kernels and TKW are explained by the pathogen spread within ears – downwards more than upwards – and the effect on yield formation which is lower for infections of the upper parts of ears. An exponential model showed high goodness of fit for gradients of infected kernels within ears (R 2 ≥ 70) except tip infection with F. culmorum. This study confirmed that FHB is a function of the primary infection site within ears. Thermography was useful to differentiate among infection scenarios and may be applied in breeding for FHB resistance.

QTL mapping of Fusarium head blight resistance in three related durum wheat populations

Key messageThe QTL Fhb1was successfully introgressed and validated in three durum wheat populations. The novel germplasm and the QTL detected will support improvement of Fusarium resistance in durum wheat.Durum wheat (Triticum durum Desf.) is particularly susceptible to Fusarium head blight (FHB) and breeding for resistance is hampered by limited genetic variation within this species. To date, resistant sources are mainly available in a few wild relative tetraploid wheat accessions. In this study, the effect of the well-known hexaploid wheat (Triticum aestivum L.) quantitative trait locus (QTL) Fhb1 was assessed for the first time in durum wheat. Three F7-RIL mapping populations of about 100 lines were developed from crosses between the durum wheat experimental line DBC-480, which carries an Fhb1 introgression from Sumai-3, and the European T. durum cultivars Karur, Durobonus and SZD1029K. The RILs were evaluated in field experiments for FHB resistance in three seasons using spray inoculation and genotyped with SSR as well as genotyping-by-sequencing markers. QTL associated with FHB resistance were identified on chromosome arms 2BL, 3BS, 4AL, 4BS, 5AL and 6AS at which the resistant parent DBC-480 contributed the positive alleles. The QTL on 3BS was detected in all three populations centered at the Fhb1 interval. The Rht-B1 locus governing plant height was found to have a strong effect in modulating FHB severity in all populations. The negative effect of the semi-dwarf allele Rht-B1b on FHB resistance was compensated by combining with Fhb1 and additional resistance QTL. The successful deployment of Fhb1 in T. durum was further substantiated by assessing type 2 resistance in one population. The efficient introgression of Fhb1 represents a significant step forward for enhancing FHB resistance in durum wheat.