A rapid and non-destructive method for spatial–temporal quantification of colonization by Pseudomonas syringae pv. tomato DC3000 in Arabidopsis and tomato

Background The bacterial leaf pathogen Pseudomonas syringae pv tomato (Pst) is the most popular model pathogen for plant pathology research. Previous methods to study the plant-Pst interactions rely on destructive quantification of Pst colonisation, which can be labour- and time-consuming and does not allow for spatial–temporal monitoring of the bacterial colonisation. Here, we describe a rapid and non-destructive method to quantify and visualise spatial–temporal colonisation by Pst in intact leaves of Arabidopsis and tomato. Results The method presented here uses a bioluminescent Pst DC3000 strain that constitutively expresses the luxCDABE operon from Photorhabdus luminescens (Pst::LUX) and requires a common gel documentation (Gel Doc) system with a sensitive CCD/CMOS camera and imaging software (Photoshop or Image J). By capturing bright field and bioluminescence images from Pst::LUX-infected leaves, we imaged the spatiotemporal dynamics of Pst infection. Analysis of bioluminescence from live Pst bacteria over a 5-day time course after spray inoculation of Arabidopsis revealed transition of the bacterial presence from the older leaves to the younger leaves and apical meristem. Colonisation by Pst:LUX bioluminescence was obtained from digital photos by calculating relative bioluminescence values, which is adjusted for bioluminescence intensity and normalised by leaf surface. This method detected statistically significant differences in Pst::LUX colonisation between Arabidopsis genotypes varying in basal resistance, as well as statistically significant reductions in Pst::LUX colonisation by resistance-inducing treatments in both Arabidopsis and tomato. Comparison of relative bioluminescence values to conventional colony counting on selective agar medium revealed a statistically significant correlation, which was reproducible between different Gel Doc systems. Conclusions We present a non-destructive method to quantify colonisation by bioluminescent Pst::LUX in plants. Using a common Gel Doc system and imaging software, our method requires less time and labour than conventional methods that are based on destructive sampling of infected leaf material. Furthermore, in contrast to conventional strategies, our method provides additional information about the spatial–temporal patterns of Pst colonisation.

Evaluation of Anthracnose Resistance in Pepper (Capsicum spp.) Genetic Resources

Anthracnose (Colletotrichum spp.), is one of the major yield losing fungal disease in both pre- and post-harvest stage of pepper (Capsicum spp.) production worldwide. Among the Colletotrichum spp., C. acutatum has strong pathogenicity, which infects both immature and mature pepper fruit leads to severe economic losses in pepper production. Inheritance of anthracnose disease resistance was evaluated with 3738 pepper genetic resources which was collected from different countries and conserved at Korean genebank. The resistance analysis against pepper anthracnose (C. acutatum) was performed on detached mature green and red fruits under laboratory conditions by spray (non-wounding) and microinjection (wounding) inoculation methods. In the primary screening, about 261 accessions were appeared to be resistant against C. acutatum in spray inoculation. The resistant accessions were further evaluated with microinjection (wounding) inoculation method using the fungal (C. acutatum) isolate of pepper anthracnose. There were highly significant differences in the disease severity and distribution of disease rating scale, considering all the sources has significant genetic variation. Finally, the anthracnose resistant pepper accessions have been validated with cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers in which, the CAPS and HRM marker analysis showed four types of genotypes such as resistant (R), susceptible (S), heterozygous (H) and Unidentified type (UT) or not detection. The Capsicum accessions showing high level of resistance to the pathogen could be used as source material in breeding programs for resistance to anthracnose disease.

Evaluating the efficacy of potential fungicide-adjuvant combinations for control of myrtle rust in New Zealand

Myrtle rust is a serious fungal disease caused by Austropuccinia psidii affecting a number of Myrtaceae species in New Zealand and elsewhere. Control with fungicides or biologicals provides a mechanism to reduce the build-up of inoculum in the short-term while other strategies are being developed or deployed for long-term management. This study evaluated the efficacy of fungicides for control of myrtle rust under controlled conditions and identified adjuvants that would promote spreading of fungicidal active ingredients across the leaf surface. The spread of fungicide on detached M. excelsa leaves was assessed by applying three different adjuvants in combination with seven fungicides. Subsequently, M. excelsa plants were treated with three fungicides/mixes, (azoxystrobin + epoxiconazole, triademinol or a natural tea-extract) at a single rate followed by inoculation with A. psidii urediniospores on day 0, 7, 14 or 21 days after spraying. The response to infection in M. excelsa plants based on different inoculation timings at days 0, 7 and 21 significantly differed among fungicide treatments. The fungicide azoxystrobin + epoxiconazole was the most effective with infections significantly lower on the adaxial leaf surface than abaxial, despite good surface coverage of fungicide being achieved on both leaf surfaces. There were significant differences among fungicides based on the proportion of infected leaves on M. excelsa plants. Day 21 post-spray inoculation indicated a significant interaction between inoculation time and fungicide on leaf disease ratings. However, this was not the case at either 28 or 35 days post-inoculation. This research contributes to fungicide options for myrtle rust control in New Zealand.

Community structure of fungal pathogens causing spikelet rot disease of naked oat from different ecological regions of China

Spikelet rot disease (SRD) is an emerging disease of the grain surface of naked oat in China that affects both grain yield and quality. The typical symptom is discoloration from the black structures of the causal fungi. Here, we investigated the fungal communities on the grain surfaces of cultivar Bayou 13 grown in ten ecological oat-producing regions of China, to identify the main pathogens of naked oat SRD. Our results showed that the growth of Alternaria spp. and Davidiella spp. exhibited a competitive relationship and was mainly affected by the elevations of all 10 ecological regions. The dominant pathogens were Davidiella spp. in Shannan Prefecture in Tibet and Haidong Prefecture in Qinghai Province and Alternaria spp. in the other eight regions. The ratios of black pathogens of interest to all pathogens in Shannan Prefecture and Haidong Prefecture were significantly lower than those of the other eight regions, thus indicating that SRD mainly occurred in regions below 2000 m (elevation). We isolated black fungal pathogens from grain surfaces and deduced that they were Alternaria spp. by sequence comparison. The blackened appearance of the grain surfaces was more evident under spray inoculation with a spore suspension of Alternaria than under the control in greenhouse experiments. The recovered pathogen was the same as the pathogen used for inoculation. We thus concluded that Alternaria alone causes naked oat SRD and mainly infects naked oat in regions below 2000 m, which provides a basis for the recognition and management of SRD of naked oat.

Assessment of inoculation techniques for screening sugarcane resistance to red stripe disease caused by Acidovorax avenae subsp. avenae

The red stripe disease caused by Acidovorax avenae subsp. avenae in sugarcane, has become a quite relevant issue in Argentina because of its high incidence in the sugarcane growing area. The resistance of host plants is the most promising method for controlling the disease. In that sense, the Estación Experimental Agroindustrial Obispo Colombres (EEAOC) has a Sugarcane Breeding Program, which generates new varieties with higher productivity and good sanitary behavior. The lack of an effective screening technique to select resistant sugarcane genotypes limits the cultivar selection process. To develop a practical and affordable method for achieving the expression of the red stripe disease, three available inoculation techniques were evaluated under controlled conditions over two sugarcane varieties, with a previously adjustment of soil composition and nutrition and relative humidity. They consisted in (i) scrubbing the leaf surface with a cotton ball soaked in the suspension of A. avenae subsp. avenae; and spraying inoculum under two conditions: (ii) leaves pre-treated with a refined sand scarification and (iii) leaves with no scarification. Fifteen plants were inoculated per cultivar and treatment according to a randomized protocol with three replicates and the severity of the disease was evaluated on a scale of 1- 9 according to the International Society of Sugarcane Technologists. The spray inoculation using a bacterial suspension of A. avenae subsp. avenae without abrasives was also field tested. These results contribute to sugarcane breeding programs, providing a tool to assess the resistance to red stripe of their materials, overcoming the lack of bacterial pressure or favorable conditions for the disease.

Characterization and Pathogenicity of Colletotrichum Species on Philodendron tatei cv. Congo in Gansu Province, China

In recent years in China, leaf spot caused by Colletotrichum species has been an emerging disease of Philodendron tatei cv. Congo. From 2016 to 2019, typical symptoms, appearing as circular or ovoid, sunken, and brown lesions with a yellow halo, were commonly observed on P. tatei cv. Congo in and around Lanzhou, Gansu Province, China. Conidiomata were often visible on infected leaf surfaces. Leaf disease incidence was approximately 5 to 20%. A total of 126 single-spored Colletotrichum isolates were obtained from leaf lesions. Multilocus phylogenetic relationships were analyzed based on seven genomic loci (ITS, ACT, GAPDH, HIS3, CAL, CHS-1, and TUB2) and the morphological characters of the isolates determined. These isolates were identified as three Colletotrichum species in this study. A further 93 isolates, accounting for 74% of all Colletotrichum isolates, were described as new species and named as Colletotrichum philodendricola sp. nov. after the host plant genus name, Philodendron; another two isolates were named as C. pseudoboninense sp. nov. based on phylogenetic and morphological relativeness to C. boninense; the other 31 isolates, belonging to the C. orchidearum species complex, were identified as a known species—C. orchidearum. Both novel species C. philodendricola and C. pseudoboninense belong to the C. boninense species complex. Pathogenicity tests by both spray and point inoculations confirmed that all three species could infect leaves of P. tatei cv. Congo. For spray inoculation, the mean infection rate of leaves on the three species was only 4.7% (0 to 12%), and the size on lesions was mostly 1 to 2 mm in length. For point inoculation, 30 days after nonwounding inoculation, the infection rate on leaves was 0 to 35%; in wounding inoculation, the infection rate of leaves was 35 to 65%; wounding in healthy leaves greatly enhanced the pathogenicity of these three species to P. tatei cv. Congo; however, the sizes of lesions among the three species were not significantly different. To our knowledge, this is the first report of Colletotrichum species associated with anthracnose diseases on P. tatei cv. Congo. Results obtained in this study will assist the disease prevention and appropriate management strategies.

Validation of Screening Technique for Cotton Bacterial Blight Resistance under Controlled Condition

Seven different methods of artificial inoculation such as 1. Carborundum injury, 2. Pin prick injury 3. Sand paper injury, 4. Syringe inoculation on lower surface of leaf without needle, 5. Syringe inoculation of veins on lower surface of leaf with needle, 6. Tooth picks inoculation on collar region and 7. Pressurized spray inoculation were evaluated to find out the efficient and precise screening method for cotton bacterial blight caused by Xanthomonas citri pv. malvacearum under controlled conditions (plant growth chamber). Inoculated seedlings were incubated at 28°C, 90% RH and 3000 LUX light intensity during day time and 22°C, 90% RH and absence of light during night time for symptom development. Among them, pin prick injury recorded maximum PDI (64.25) in 20-24 days post inoculation followed by sand paper injury (56.50 PDI) in 23-27 days post inoculation on 20 day old LRA 5166 cotton seedlings compared to other methods. Both these methods developed all types of symptoms. Initial symptom of water soaked lesion was appeared in 7-8 days post inoculation in pin prick injury while it was 9-10 days in sand paper injury.