Plant Science

Plant growth–promoting rhizobacteria (PGPR) can be used as biofertilizers to enhance crop growth for better yield and soil fertility restoration. PGPR possesses certain traits such as nutrient solubilization, phytohormone production, and production of key enzymes for improved crop growth. These traits are also important for inhibiting the growth of plant root pathogens, improving root development, and conferring stress tolerance. However, the mere presence of PGPR traits in isolated bacteria may not directly reflect an improvement in plant growth, warranting researchers to evaluate phenotypic and physiological changes upon inoculation. The current manuscript provides a detailed step-by-step procedure for inoculating the PGPR Staphylococcus sciuri into seeds and seedlings of rice and tomato plants for visualizing the enhancement of root and shoot growth. The surface-sterilized seeds of rice and tomato plants are inoculated overnight with an actively grown log-phase culture of S. sciuri, and differences in growth and biomass of seedlings that emerged from the inoculated and uninoculated seeds are analyzed 10 days after germination. Plants grown in pots with sterile soil are also treated with PGPR S. sciuri by soil drenching. A remarkable increase in root and shoot growth is observed in inoculated plants. We suggest that treating seeds with bacteria and enriching the soil with bacterial inoculum provides an adequate load of PGPR that facilitates growth improvement. This method can be a reliable choice for screening and evaluating plant growth promotion by either isolated bacteria or bacterial consortia with plant-beneficial traits.

Antimicrobial peptides are effective agents against various pathogens, often targeting essential processes like protein translation to exert their antimicrobial effects. Traditional methods such as puromycin labeling have been extensively used to measure protein synthesis in mammalian and yeast systems; however, protocols tailored for plant pathogenic filamentous fungi, particularly those investigating translation inhibition by antifungal peptides, are lacking. This protocol adapts puromycin labeling to quantify translation inhibition in Botrytis cinerea germlings treated with antifungal peptides. Optimizing the method specifically for fungal germlings provides a precise tool to investigate peptide effects on fungal protein synthesis, advancing our understanding of translation dynamics during pathogen–host interactions in filamentous fungi.

In nature, filamentous fungi interact with plants. These fungi are characterized by rapid growth in numerous substrates and under minimal nutrient requirements. Investigating the interaction of these fungi with their plant hosts under controlled conditions is of importance for many researchers aiming to proceed with molecular or microscopical investigations of their favorite plant–fungus interaction system. The speed of growth of these fungi complicates transferring plant–fungal interaction systems in laboratory conditions. The issue is more complicated when monoxenic conditions are desired, to ensure that only two members (a fungus and a plant) are present in the system under study. Here, two simple closed systems for investigating plant–filamentous fungi associations under laboratory, monoxenic conditions are described, along with their limitations. The plant and fungal growth conditions, methods for sampling, staining, sectioning, and subsequent microscopical imaging of colonized plant tissues with affordable, common laboratory tools are described.

The Fusarium genus includes various fungi of great significance in agriculture. Fusarium solani f. sp. eumartii (F. eumartii), traditionally known as a potato pathogen, has also been identified as a cause of disease in tomatoes. This protocol provides a detailed, efficient, and robust flood-inoculation method for assessing F. eumartii infection of young tomato seedlings grown on MS medium plates. It includes the evaluation of the lesion area and the quantification of the remaining fungal inoculum in tomato seedlings. In summary, the straightforwardness and efficiency of this bioassay make it a powerful quantitative tool for selecting fungicidal compounds or defense response inducers in tomato plants, offering a promising approach with significant potential for preventing fungal diseases in crops.

The root parasitic weed Striga hermonthica has a devastating effect on sorghum and other cereal crops in Sub-Saharan Africa. Available Striga management strategies are rarely sufficient or not widely accessible or affordable. Identification of soil- or plant-associated microorganisms that interfere in the Striga infection cycle holds potential for development of complementary biological control measures. Such inoculants should be preferably based on microbes native to the regions of their application. We developed a method to assess microbiome-based soil suppressiveness to Striga with a minimal amount of field-collected soil. We previously used this method to identify the mechanisms of microbe-mediated suppression of Striga infection and to test individual microbial strains. Here, we present protocols to assess the functional potential of the soil microbiome and individual bacterial taxa that adversely affect Striga parasitism in sorghum via three major known suppression mechanisms. These methods can be further extended to other Striga hosts and other root parasitic weeds.

The roots of herbaceous and woody plants growing in soil are complex structures that are affected by both natural and artificial fungal colonization to various extents. To obtain comprehensive information about the overall distribution of fungi or oomycetes inside a plant root system, rapid, effective, and reliable screening methods are required. To observe both fine roots, i.e., a common site for penetration of fungi and oomycetes, and mature roots, different techniques are required to overcome visual barriers, such as root browning or tissue thickening. In our protocol, we propose using fast, cost-effective, and non-harmful methods to localize fungal or oomycete structures inside plant roots. Root staining with a fluorescent dye provides a quick initial indication of the presence of fungal structures on the root surfaces. The protocol is followed by clearing and staining steps, resulting in a deeper insight into the root tissue positioning, abundance, and characteristic morphological/reproductive features of fungal or oomycete organisms. If required, the stained samples can be prepared by using freeze-drying for further observations, including advanced microscopic techniques.

Maize is one of the most important crops in the world, and ensuring its successful growth and productivity is crucial for global food security. One way to enhance maize growth and productivity is by improving the colonization of its roots by beneficial microorganisms. In this regard, Serendipita indica, a plant growth–promoting fungus, has gained attention for its ability to enhance plant growth and productivity, especially in cereal crops and medicinal plants. Previous studies have shown that S. indica can colonize various plant species, including maize, but the efficiency of the colonization process in maize seedlings has not been extensively characterized. This protocol outlines a method for efficient colonization of maize seedlings with the beneficial fungus S. indica. The protocol includes the preparation of stock solutions, maintenance and growth of S. indica, surface sterilization and germination of seeds, preparation of S. indica chlamydospores, and colonization of maize plants with S. indica. The advantages of this protocol include the use of surface sterilization techniques that minimize contamination, the production of a large number of viable chlamydospores, and efficient colonization of maize seedlings with S. indica. This protocol may be useful for researchers studying the role of S. indica in promoting plant growth and combating biotic and abiotic stress. Additionally, this protocol may be used in the development of biofertilizers using S. indica as a means of increasing crop yields and reducing dependence on synthetic fertilizers. Overall, this protocol offers a reliable and efficient method for colonizing maize seedlings with S. indica and may have potential applications in the agricultural industry. This study also provides a valuable tool for researchers interested in studying plant–microbe interactions in maize and highlights the potential of S. indica as a biocontrol agent to enhance maize productivity under adverse conditions.

Key features

• This protocol builds upon the method developed by Narayan et al. (2022), and its application optimized for the root endophytic symbiotic fungus S. indica.

• This protocol also allows for histochemical analysis to visualize the colonized fungal spores in the root cells of host plant species.

• This protocol helps in mathematical calculation of the percent colonization or efficiency of colonization.

• This protocol utilizes readily available laboratory equipment, including a light microscope, autoclave, and laminar flow hood, ensuring ease of reproducibility in other research laboratories.

Graphical overview

Strawberries are delicious and nutritious fruits that are widely cultivated and consumed around the world, either fresh or in various products such as jam, juice, and ice cream. Botrytis cinerea is a fungal pathogen that causes gray mold disease on many crops, including strawberries. Disease monitoring is an important aspect for growing commercial crops like strawberry because there is an urgent need to develop effective strategies to control this destructive gray mold disease. In this protocol, we provide an important tool to monitor the gray mold fungal infection progression in different developmental stages of strawberry. There are different types of inoculation assays for B. cinerea on strawberry plants, such as in vitro (in/on a culture medium) or in vivo (in a living plant). In vivo inoculation assays can be performed at early, middle, and late stages of strawberry development. Here, we describe three methods for in vivo inoculation assays of B. cinerea on strawberry plants. For early-stage strawberry plants, we modified the traditional fungal disc inoculation method to apply to fungal infection on strawberry leaves. For middle-stage strawberry plants, we developed the flower infection assay by dropping fungal conidia onto flowers. For late-stage strawberry plants, we tracked the survival rate of strawberry fruits after fungal conidia infection. This protocol has been successfully used in both lab and greenhouse conditions. It can be applied to other flowering plants or non-model species with appropriate modifications.

Key features

• Fungal disc inoculation on early-stage strawberry leaves.

• Fungal conidia inoculation on middle-stage strawberry flowers.

• Disease rating for late-stage strawberry fruits.

• This protocol is applicable to the other flowering plants with appropriate modifications.

Graphical overview

In vivo infection progression assays of gray mold fungus Botrytis cinerea at different developmental stages of strawberry. Created with BioRender.com.

Barley (Hordeum vulgare) is one of the most important agricultural crops in the world, but pathogen infections regularly limit its annual yield. A major threat is the infection with the biotrophic leaf rust fungus, Puccinia hordei. Rust fungi have a complex life cycle, and existing resistances can be easily overcome. To address this problem, it is crucial to develop barley varieties with improved and durable resistance mechanisms. An essential step towards this goal is a simple and reproducible infection protocol to evaluate potential resistance phenotypes in the lab. However, available protocols sometimes lack detailed procedure or equipment information, use spore application methods that are not suitable for uniform spore dispersion, or require special mineral oils or engineered fluids. In addition, they are often optimized for pathogen-dedicated greenhouses or phytochambers, which may not be available to every research institute. Here, we describe an easy and user-friendly procedure to infect barley with Puccinia hordei on a small laboratory scale. This procedure utilizes inexpensive and simple tools to evenly split and apply spores to barley leaves. The treated plants are incubated in affordable and small phytocabinets. Our protocol enables a quick and reproducible infection of barley with leaf rust, a method that can easily be transferred to other rust fungi, including stripe rust, or to other plant species.

Key features

• Step-by-step infection protocol established for barley cv. Golden Promise, the gold standard genotype for genetic transformation

• Plant age–independent protocol

• Precise spore application by using inexpensive pipe cleaners for uniform symptom formation and increased reproducibility

• No specialized equipment needed

• Includes simple spore harvesting method

• Protocol is applicable to other biotrophic pathogens (stripe rust or powdery mildew) and other plants (e.g., wheat)

• Protocol is also applicable for a detached leaf assay

Graphical overview

Arabidopsis thaliana-Pseudomonas syringae pathosystem has been used as an important model system for studying plant-microbe interactions, leading to many milestones and breakthroughs in the understanding of plant immune system and pathogenesis mechanisms. Bacterial infection and plant disease assessment are key experiments in the studies of plant-pathogen interactions. The hypersensitive response (HR), which is characterized by rapid cell death and tissue collapse after inoculation with a high dose of bacteria, is a hallmark response of plant effector-triggered immunity (ETI), one layer of plant immunity triggered by recognition of pathogen-derived effector proteins. Here, we present a detailed protocol for bacterial disease and hypersensitive response assays applicable to studies of Pseudomonas syringae interaction with various plant species such as Arabidopsis, Nicotiana benthamiana, and tomato.