Differential root and cell regulation of maize aquaporins by the arbuscular mycorrhizal symbiosis highlights its role in plant water relations.

This study aims to elucidate if the regulation of plant aquaporins by the arbuscular mycorrhizal (AM) symbiosis occurs only in roots or cells colonized by the fungus or at whole root system. Maize plants were cultivated in a split-root system, with half of the root system inoculated with the AM fungus and the other half uninoculated. Plant growth and hydraulic parameters were measured and aquaporin gene expression was determined in each root fraction and in microdissected cells. Under well-watered conditions, the non-colonized root fractions of AM plants grew more than the colonized root fraction. Total osmotic and hydrostatic root hydraulic conductivities (Lo and Lpr) were higher in AM plants than in non-mycorrhizal plants. The expression of most maize aquaporin genes analysed was different in the mycorrhizal root fraction than in the non-mycorrhizal root fraction of AM plants. At the cellular level, differential aquaporin expression in AM-colonized cells and in uncolonized cells was also observed. Results indicate the existence of both, local and systemic regulation of plant aquaporins by the AM symbiosis and suggest that such regulation is related to the availability of water taken up by fungal hyphae in each root fraction and to the plant need of water mobilization.

First Report of Shoot Blight of Grapevine Caused by Sclerotinia sclerotiorum in Illes Balears, Mallorca, Spain.

In 2021, grapevines (Vitis vinifera L.) cv. Callet growing in a commercial vineyard located at Pollença (northeast of the island of Majorca, Spain) showed severe symptoms of shoot blight during spring and early summer, with an incidence of 70%. Symptoms consisted of elongated cankered-like lesions, surrounded by water-soaked darker tissues, that developed at the base or around the middle nodes of the shoot. For fungal isolation, shoot samples with lesions were collected, surface disinfected with 2% NaCl for 90s, rinsed twice with deionized water and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25°C under 12 h light-darkness for 6 days. Isolations consistently yielded on kind of fungal colonies that produced white mycelium and black spherical to elongated sclerotia (2 to 10 mm in diameter). Morphological characterization was consistent with the description of Sclerotinia sclerotiorum (Lib.) de Bary (Bolton et al. 2006). Three isolates (UIB 118-1, UIB 118-26, and UIB 129-41) were preserved and deposited in the Culture Collection of Microbiology-Faculty of Sciences, University of Balearic Islands, Spain. Genomic DNA was extracted from isolates UIB 118-26 and UIB 129-41 using the EZNA Miniprep Kit (Omega Bio-Tek, Norcross, GA). The internal transcribed spacer (ITS) region of ribosomal DNA, ß-tubulin (BTUB) and calmodulin (CAL) gene regions were amplified using ITS1F-ITS4 (Gardes and Bruns, 1996; White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995) and CAL228F/CAL737R (Carbone and Kohn 1999) primer sets, respectively. Amplicons were sequenced and deposited in GenBank with accession numbers MZ604647 and MZ604648 for ITS, OK634402 and OK634403 for BTUB and OK634404 and OK634405 for CAL. BLASTn search showed that isolates were >99 % (ITS, BTUB and CAL) identical to S. sclerotiorum GenBank accession no. KF859933, CP017815 and KF871381, respectively. Pathogenicity tests were conducted using eight one-year old grapevines cv. Cabernet Sauvignon. Old and new green shoots were inoculated by inserting a 6-mm plug of mycelium taken from actively growing cultures on PDA into cuts made at the base and at the distal part of each shoot with a sterile scalpel with a total of eight inoculation points per plant. Inoculated wounds were sealed with Parafilm tape to avoid rapid dehydration. Inoculated plants and an equal number of wounded but non-inoculated plants (negative controls) were maintained at 25 ± 1°C for 48 h in plastic containers to ensure a high relative humidity (>90%). After 5 days, the infection girdled and rotted the green new shoots, whereas the older partially lignified shoots developed a localized long brown lesion that reached 16 cm in length. Due to the rotting of the basal part of the petiole, leaves turned gray, wilted, and died, easily detaching from the stem. In advanced stages of the disease, 7 days after infection, branches died and fell with the leaves remained attached (Fig 1 A, B). Reisolations from diseased shoots were successfully performed on PDA to fulfill Koch's postulates. S. slerotiorum was previously reported on grapevine causing shoot blight in Chile (Latorre and Guerrero, 2001), Korea (Jong-Han et al. 2009), California-USA (Boland and Hall, 1994) and Australia (Hall et al. 2002). AlsoS. sclerotiorum was reported among the endophytic mycobiota associated with Vitis vinifera in the Iberian Peninsula (Gonzalez and Tello, 2011) but not as a pathogen causing visible symptoms on that crop. So, this is the first report of the occurrence of S. slerotiorum as a pathogen of grapevines in Spain causing symptoms of canker and shoot blight. This finding highlights a potential risk of this fungal disease for the wine industry in the Mediterranean region and specially for Spain, the country with the largest acreage devoted to grapevines. Although chemical and biological are suitable control strategies, disease management is difficult as sclerotia of Sclerotinia can remain in the soil for up to eight years (Adams and Ayears, 1979), and preventive surveys are greatly recommended as an important epidemiological tool to monitor the epidemiology of disease and identify potential outbreaks of this new pathogen on grapevine in Spain.

Dual Inoculation with Rhizophagus irregularis and Bacillus megaterium Improves Maize Tolerance to Combined Drought and High Temperature Stress by Enhancing Root Hydraulics, Photosynthesis and Hormonal Responses.

Climate change is leading to combined drought and high temperature stress in many areas, drastically reducing crop production, especially for high-water-consuming crops such as maize. This study aimed to determine how the co-inoculation of an arbuscular mycorrhizal (AM) fungus (Rhizophagus irregularis) and the PGPR Bacillus megaterium (Bm) alters the radial water movement and physiology in maize plants in order to cope with combined drought and high temperature stress. Thus, maize plants were kept uninoculated or inoculated with R. irregularis (AM), with B. megaterium (Bm) or with both microorganisms (AM + Bm) and subjected or not to combined drought and high temperature stress (D + T). We measured plant physiological responses, root hydraulic parameters, aquaporin gene expression and protein abundances and sap hormonal content. The results showed that dual AM + Bm inoculation was more effective against combined D + T stress than single inoculation. This was related to a synergistic enhancement of efficiency of the phytosystem II, stomatal conductance and photosynthetic activity. Moreover, dually inoculated plants maintained higher root hydraulic conductivity, which was related to regulation of the aquaporins ZmPIP1;3, ZmTIP1.1, ZmPIP2;2 and GintAQPF1 and levels of plant sap hormones. This study demonstrates the usefulness of combining beneficial soil microorganisms to improve crop productivity under the current climate-change scenario.

A non-K+-solubilizing PGPB (Bacillus megaterium) increased K+ deprivation tolerance in Oryza sativa seedlings by up-regulating root K+ transporters.

Potassium is one of the principal macronutrients required by all plants, but its mobility is restricted between soil compartments. Numerous studies have shown that Plant Growth Promoting Bacteria (PGPB) can facilitate nutrient uptake. The present work examined the effects of the PGPB (Bacillus megaterium) on rice plants subjected to potassium deprivation. To study only direct effects of B. megaterium, we first checked its lack of capacity to solubilize soil K. Rice plants were provided with 1.5 mM K (100%) or 0.015 mM K (1%) and growth related parameters, nutrient concentrations and gene expression of K+ transporters were determined. After two weeks, the 1% K treatment reduced growth of non-inoculated plants by about 50% compared with the 100% K treatment. However, there was no effect of reduced K nutrition on growth of inoculated plants. The reduction in growth in non-inoculated plants was accompanied by a similar reduction in K+ concentration in both roots and leaves and an overall 80% reduction of the plant potassium concentrations. In inoculated plants a 50% reduction occurred only in leaves. The expression of the K+ transporters HKT1;1, 1;2, 1;5, 2;2, 2;3 and 2;4 was up-regulated by the inoculation of B. megaterium under K deprivation conditions, explaining their higher K tissue concentrations and growth. Thus, the bacterial strain improved plant potassium nutrition without affecting K+ availability in the soil. The results demonstrate the potential of this bacteria for using as a biofertilizer to reduce the amount of potassium fertilizers to be applied in the field.

In Vivo Metabolic Regulation of Alternative Oxidase under Nutrient Deficiency-Interaction with Arbuscular Mycorrhizal Fungi and Rhizobium Bacteria.

The interaction of the alternative oxidase (AOX) pathway with nutrient metabolism is important for understanding how respiration modulates ATP synthesis and carbon economy in plants under nutrient deficiency. Although AOX activity reduces the energy yield of respiration, this enzymatic activity is upregulated under stress conditions to maintain the functioning of primary metabolism. The in vivo metabolic regulation of AOX activity by phosphorus (P) and nitrogen (N) and during plant symbioses with Arbuscular mycorrhizal fungi (AMF) and Rhizobium bacteria is still not fully understood. We highlight several findings and open questions concerning the in vivo regulation of AOX activity and its impact on plant metabolism during P deficiency and symbiosis with AMF. We also highlight the need for the identification of which metabolic regulatory factors of AOX activity are related to N availability and nitrogen-fixing legume-rhizobia symbiosis in order to improve our understanding of N assimilation and biological nitrogen fixation.

Arbuscular Mycorrhizal Fungi Confer Salt Tolerance in Giant Reed (Arundo donax L.) Plants Grown Under Low Phosphorus by Reducing Leaf Na+ Concentration and Improving Phosphorus Use Efficiency.

Salinization is one of the major causes of agricultural soil degradation worldwide. In arid and semi-arid regions with calcareous soils, phosphorus (P) deficiency further worsens the quality of salinized soils. Nonetheless, nutrient poor soils could be suitable of producing second-generation energy crops. Due to its high biomass production, Arundo donax L. (giant reed) is one of the most promising species for energy and second-generation biofuel production. A. donax can be propagated by micropropagation, an in vitro technique that produces high number of homogeneous plantlets. However, crop establishment is often compromised due to poor plantlet acclimatization to the soil environment. Arbuscular mycorrhizal fungi (AM) are components of soil-plant systems able to increase root phosphorus uptake and to confer the plant an increase tolerance to salinity with a consequent enhancement effect of plant growth and yield. In the present study, the relative importance of the early symbiosis establishment between AM fungi and A. donax micropropagated plantlets in the response to salt stress under low phosphorus availability was determined. A commercial inoculum which contained two different AM fungi species: Rhizophagus intraradices and Funneliformis mosseae was used. AM-symbionts (AM) and non-symbionts plants were grown at two phosphorus [2.5 µM (C) and 0.5 mM (P)] and three NaCl (1, 75 and 150 mM) concentrations in a room chamber under controlled conditions. After 5 weeks, AM root colonization was 60, 26 and 15% in 1, 75 and 150 mM NaCl-treated plants, respectively. At 1 and 75 mM NaCl, AM plants showed increased growth. In all saline treatments, AM plants had decreased Na+ uptake, Na+ root-to-shoot translocation, Na+/K+ ratio and increased P and K use efficiencies with respect to C and P plants. AM improved the nutritional status of A. donax plants by enhancing nutrient use efficiency rather than nutrient uptake. Increased phosphorus use efficiency in AM plants could have benefited ion (Na+ and K+) uptake and/or allocation and ultimately ameliorate the plant's response to saline conditions.

Phosphorus concentration coordinates a respiratory bypass, synthesis and exudation of citrate, and the expression of high-affinity phosphorus transporters in Solanum lycopersicum.

Plants exhibit respiratory bypasses (e.g., the alternative oxidase [AOX]) and increase the synthesis of carboxylates in their organs (leaves and roots) in response to phosphorus (P) deficiency, which increases P uptake capacity. They also show differential expression of high-affinity inorganic phosphorus (Pi) transporters, thus avoiding P toxicity at a high P availability. The association between AOX and carboxylate synthesis was tested in Solanum lycopersicum plants grown at different soil P availability, by using plants grown under P-sufficient and P-limiting conditions and by applying a short-term (24 hr) P-sufficient pulse to plants grown under P limitation. Tests were also performed with plants colonized with arbuscular mycorrhizal fungi, which increased plant P concentration under reduced P availability. The in vivo activities of AOX and cytochrome oxidase were measured together with the concentration of carboxylates and the P concentration in plant organs. Gene transcription of Pi transporters (LePT1 and LePT2) was also studied. A coordinated response between plant P concentration with these traits was observed, indicating that a sufficient P availability in soil led to a suppression of both AOX activity and synthesis of citrate and a downregulation of the transcription of genes encoding high-affinity Pi transporters, presumably to avoid P toxicity.

Respiratory ATP cost and benefit of arbuscular mycorrhizal symbiosis with Nicotiana tabacum at different growth stages and under salinity.

Growth and maintenance partly depend on both respiration and ATP production during oxidative phosphorylation in leaves. Under stress, ATP is needed to maintain the accumulated biomass. ATP production mostly proceeds from the cytochrome oxidase pathway (COP), while respiration via the alternative oxidase pathway (AOP) may decrease the production of ATP per oxygen consumed, especially under phosphorus (P) limitation and salinity conditions. Symbiosis with arbuscular mycorrhizal (AM) fungi is reputed by their positive effect on plant growth under stress at mature stages of colonization; however, fungal colonization may decrease plant growth at early stages. Thus, the present research is based on the hypothesis that AM fungus colonization will increase both foliar respiration and ATP production at mature stages of plant growth while decreasing them both at early stages. We used the oxygen-isotope-fractionation technique to study the in vivo respiratory activities and ATP production of the COP and AOP in AM and non-AM (NM) tobacco plants grown under P-limiting and saline conditions in sand at different growth stages (14, 28 and 49days). Our results suggest that AM symbiosis represents an ATP cost detrimental for shoot growth at early stages, whilst it represents a benefit on ATP allowing for faster rates of growth at mature stages, even under salinity conditions.

Arbuscular Mycorrhizal Symbiosis with Arundo donax Decreases Root Respiration and Increases Both Photosynthesis and Plant Biomass Accumulation.

The effect of arbuscular mycorrhiza (AM) symbiosis on plant growth is associated with the balance between costs and benefits. A feedback regulation loop has been described in which the higher carbohydrate cost to plants for AM symbiosis is compensated by increases in their photosynthetic rates. Nevertheless, plant carbon balance depends both on photosynthetic carbon uptake and respiratory carbon consumption. The hypothesis behind this research was that the role of respiration in plant growth under AM symbiosis may be as important as that of photosynthesis. This hypothesis was tested in Arundo donax L. plantlets inoculated with Rhizophagus irregularis and Funneliformis mosseae. We tested the effects of AM inoculation on both photosynthetic capacity and in vivo leaf and root respiration. Additionally, analyses of the primary metabolism and ion content were performed in both leaves and roots. AM inoculation increased photosynthesis through increased CO2 diffusion and electron transport in the chloroplast. Moreover, respiration decreased only in AM roots via the cytochrome oxidase pathway (COP) as measured by the oxygen isotope technique. This decline in the COP can be related to the reduced respiratory metabolism and substrates (sugars and tricarboxylic acid cycle intermediates) observed in roots.