Inoculation methods and agressiveness of Macrophomina phaseolina isolates in cowpea

ABSTRACT: The fungi Macrophomina phaseolina is the charcoal rot causal agent, one of the most important cowpea crop disease in semiarid regions can causes 100% yield losses. The search for resistant genotypes requires efficient phenotyping. In addition, there is the problem of great variation in aggressiveness between isolates. This study aimed to 1) test three methods of inoculation in semiarid conditions, and 2) to evaluate the aggressiveness of isolates of M. phaseolina. In the first experiment carried out in greenhouse, the inoculations methods were evaluated, using two cowpea lines, three inoculation methods and three pathogen isolates. On the second experiment, fifteen M. phaseolina isolates were inoculated in one cultivar to evaluate their aggressiveness. By assessing the length of the lesions and the severity of the disease using an index, we identified the toothpick inoculation method as the most efficient. Toothpick method allowed to discriminate the genotypes and the aggressiveness of the pathogen.

Establishment of an Artificial Inoculation Method of Ustilaginoidea virens without Damaging the Rice Panicle Sheaths

Rice false smut (RFS) is a destructive disease of rice worldwide caused by Ustilaginoidea virens. There is a lack of efficient and stable artificial inoculation method to simulate the natural infection of U. virens, which is an important factor limiting further research on the disease. The purpose of this study was to establish an artificial inoculation method, which can simulate the natural infection process of U. virens without destroying the panicle sheath structure of rice. In this research, rice plants were inoculated by soaking roots at the seedling stage, spraying at the tillering stage, injecting at the booting stage, and again spraying at the flowering stage to determine the appropriate artificial inoculation time. The panicle sheath instillation method and injection inoculation method were compared. The results show that stages 6 to 8 of young panicle differentiation are an important period for U. virens infection. There were no significant differences in the mean rates of infected panicles, mean rates of infected grains, and maximum infected grains per panicle between the two inoculation methods. However, the frequency of RFS ball occurrence at the upper part of the panicles was significantly higher on the spikelets inoculated by the injection method than that of spikelets inoculated by natural infection and panicle sheath instillation. Therefore, panicle sheath instillation method was more similar to the natural infection of U. virens in the field. This research exhibited an innovative artificial inoculation method for identification of U. virens pathogenicity and evaluation of rice resistance against RFS.

Inoculum Level and Inoculation Method Influences on the Pathogenic Activities of Meloidogyne incognita in Studied Model Plant Okra (Abelmoschus esculentus L. Moench)

Agricultural activities such as watering crops with nematode-infested water from wells and boreholes, and using infected plant debris as manure or mulch increase root-knot nematode infection. So, this study aims at assessing the influence of the inoculation method and inoculum level of Meloidogyne incognita on the development of root galls on okra plants. Two M. incognita inoculation methods (suspension of individuals and galled root explants) and six inoculum levels (0, 10, 100, 500, 1000 and 2000 second-stage larvae/plant) were studied. The gall index, total numbers and reproductive factor of M. incognita were used to assess the effect of treatments on root gall development. Unlike the reproductive factor, gall index and the total numbers of M. incognita increased with their inoculum level. The pathogenic activities of M. incognita were most significant when crop soils were infested with galled root explants. However, an inverse relationship was found between the inoculum levels of M. incognita and the okra plant’s development. It is reflected by negative correlation coefficients ranging from -0.90 to -0.62. It is therefore important to burn roots infected with root-knot nematodes left in fields so that they do not act as an inoculum for crops.

First Report of a Phytopythium litorale Species Causing Crown and Root Rot on Rhododendron pulchrum in China

During a survey of pathogenic oomycetes in Nanjing, China from June 2019 to October 2020, at least ten adjacent Rhododendron pulchrum plants at a Jiangjun Mountain scenic spot showed symptoms of blight, and crown and root discoloration . Symptomatic root tissues collected from three 6-year-old plants were rinsed with water, cut into 10-mm pieces, surface sterilized with 70% ethanol for 1 min, and plated onto 10% clarified V8 PARP agar (cV8A-PARP) containing pimaricin (20 mg/liter), ampicillin (125 mg/liter), rifampicin (10 mg/liter), and pentachloronitrobenzene (20 mg/liter). Four Pythium-like isolates were recovered after three days of incubation at 26°C, and purified using hyphal-tipping. Ten agar plugs (2×2 mm2) of each isolate were grown in 10 mL of 10% clarified V8 juice (cV8) in a 10 cm plate at 26°C for 3 days to produce mycelial mats, and then the cV8 was replaced with sterile water. To stimulate sporangial production, three to five drops of soil extract solution were added to each plate. Sporangia were terminal, ovoid to globose, and the size is 24 to 45.6 (mean 34.7) (n=10.8) in length x 23.6 to 36.0 (mean 29.8) (n=6.2) in width. Gametangia were not observed in cV8A or liquid media after 30 days. For colony morphology, the isolates were sub-cultured onto three solid microbial media (cV8A-PARP, potato dextrose agar, corn meal agar) . All isolates had identical morphological features in the three media. Complete ITS and partial LSU and cox2 gene regions were amplified using primer pairs ITS1/ITS4, NL1/NL4, and FM58/FM66 , respectively. The ITS, LSU, and cox2 sequences of isolate PC-dj1 (GenBank Acc. No. MW205746, MW208002, MW208003) were 100.00% (936/936 nt), 100.00% (772/772 nt), and 99.64% (554/556 nt) identical to those of JX985743, MT042003, and GU133521, respectively. We built a maximum-likelihood tree of Phytopythium species using the concatenated dataset (ITS, LSU, cox2) to observe interspecific differences. Based on the morphological characters and sequences, isolate PC-djl was identified as Phytopythium litorale . As the four isolates (PC-dj1, PC-dj2, PC-dj3 and PC-dj4) tested had identical morphological characters and molecular marker sequences, the pathogenicity of the representative isolate, PC-dj1, was tested using two inoculation methods on ten one-year-old R. pulchrum plants. For the first inoculation method, plants were removed from the pot, and their roots were rinsed with tap water to remove the soil. Each of these plants was placed in a glass flask containing 250 mL of sterile water and 10 blocks (10 x 10 mm2) of mycelial mats harvested from a three-day-old culture of P. litorale, while the other plant was placed in sterile water as a control, and incubated at 26°C. After three days, symptoms including crown rot, root rot and blight was observed on the inoculated plants whereas the control remained asymptomatic. For the second inoculation method, ten plants were dug up to expose the root ball. Ten three-day-old cV8A plugs (5×5 mm2) from a PC-dj1 culture or sterile cV8A plugs were evenly insert into the root ball of a plant before it was planted back into the original pots. Both plants were maintained in a growth chamber set at 26°C with a 12/12 h light/dark cycle and irrigated as needed. After 14 to 21 days, the inoculated plant had symptoms resembling those in the field , while the control plant remained asymptomatic. Each inoculation method was repeated at triplicate and the outcomes were identical. Phytopythium isolates with morphological features and sequences identical to those of PC-dj1 were recovered from rotted crown and root tissues of all inoculated plants. Previously, P. litorale was found causing diseases of apple and Platanus orientalis in Turkey, fruit rot and seedling damping-off of yellow squash in southern Georgia, USA. This is the first report of this species causing crown and root rot on R. pulchrum, an important ornamental plant species in China. Additional surveys are ongoing to determine the distribution of P. litorale in the city of Nanjing.

Corneal Culture in Infectious Keratitis: Effect of the Inoculation Method and Media on the Corneal Culture Outcome

Background: To compare two different methods of corneal culture in infectious keratitis: multiple sampling for direct inoculation and enrichment (standard method) and a single sample via transport medium for indirect inoculation (indirect inoculation method). Methods: Prospective inclusion of patients fulfilling predefined criteria of infectious keratitis undergoing corneal culture according to both studied methods in a randomized order. Results: The standard method resulted in a significantly higher proportion of positive culture outcomes among the 94 included episodes of infectious keratitis (61%; 57/94) than the indirect inoculation method (44%; 41/94) (p = 0.002) and a significantly higher proportion of microorganisms than the indirect inoculation method, with a Cohen’s kappa of 0.38 (95% CI: 0.28–0.49) for agreement between the methods. Subanalysis of culture results showed that direct inoculation on gonococcal agar only combined with the indirect inoculation method resulted in a similar rate of culture positive patients and proportion of detected microorganisms to the standard method. Conclusion: Indirect inoculation of one corneal sample cannot replace direct inoculation of multiple corneal samples without loss of information. A combination of directly and indirectly inoculated samples can reduce the number of corneal samples by four without statistically significant differences in culture outcome or in the proportion of detected microorganisms.

Antibacterial activity testing methods for hydrophobic patterned surfaces

One strategy to decrease the incidence of hospital-acquired infections is to avoid the survival of pathogens in the environment by the development of surfaces with antimicrobial activity. To study the antibacterial behaviour of active surfaces, different approaches have been developed of which ISO 22916 is the standard. To assess the performance of different testing methodologies to analyse the antibacterial activity of hydrophobic surface patterned plastics as part of a Horizon 2020 European research project. Four different testing methods were used to study the antibacterial activity of a patterned film, including the ISO 22916 standard, the immersion method, the touch-transfer inoculation method, and the swab inoculation method, this latter developed specifically for this project. The non-realistic test conditions of the ISO 22916 standard showed this method to be non-appropriate in the study of hydrophobic patterned surfaces. The immersion method also showed no differences between patterned films and smooth controls due to the lack of attachment of testing bacteria on both surfaces. The antibacterial activity of films could be demonstrated by the touch-transfer and the swab inoculation methods, that more precisely mimicked the way of high-touch surfaces contamination, and showed to be the best methodologies to test the antibacterial activity of patterned hydrophobic surfaces. A new ISO standard would be desirable as the reference method to study the antibacterial behaviour of patterned surfaces.

Basal Rachis Internode Injection (BRII): A novel inoculation method to evaluate wheat resistance to Fusarium head blight

Fusarium head blight (FHB) is one of the most destructive fungal diseases of wheat. Difficulties in reliably phenotyping of this disease, however, greatly hindered the understanding of the mechanism of wheat-pathogen interaction and genetic improvement of FHB resistance. Here we report a novel inoculation method called “Basal Rachis Internode Injection” (BRII), which is implemented by injecting inoculum into the basal internode of a rachis instead of a floret as done in single floret inoculation (SFI). One of the prominent advantages of BRII over SFI and other traditional methods lies in its independence of moisture-maintaining system that is required for all existing methods, thus being insensitive to environmental humidity and cost-effective. Another unique feature for BRII is that this method produces nearly clear-cut reaction types, by which FHB resistance could be treated as a qualitative trait because generally no FHB symptom appeared on the spikelets of resistant genotypes. In addition, BRII outperformed SFI by higher infection rate and better goodness-of-fit with known FHB resistance and QTL components in a panel of 15 genotypes, as well as two populations of recombinant inbred lines (RILs) segregating in Fhb1. To be noteworthy, BRII and SFI methods are not mutually replaceable but rather complimentary to each other since each method has its own advantage in differentiating FHB resistance among genotypes. Combining these two methods would significantly improve the reliability and consistency of FHB phenotyping in wheat.