Different patterns in root and soil fungal diversity drive plant productivity of the desert truffle <i>Terfezia claveryi</i> in plantation

SummaryThe desert truffle Terfezia claveryi is one of the few mycorrhizal fungi currently in cultivation in semiarid and arid areas. Agroclimatic parameters seem to affect its annual yield, but there is no information on the influence of biotic factors. In this study, fungal diversity was analysed by high‐throughput sequencing of the ITS2 rDNA region from soil and root samples to compare productive and non‐productive mycorrhizal plants in a 4‐years old plantation (Murcia, Spain). The fungal metaprofile was dominated by Ascomycota phylum. Desert truffle productivity was driven by different patterns of fungal species composition in soil (species replacement) and root (species richness differences). Moreover, positive associations for ectomycorrhizal and negative for arbuscular mycorrhizal guilds were found in productive roots, and positive associations for fungal parasite‐plant pathogen guild in non‐productive ones. Soil samples were dominated by pathotroph and saprotroph trophic modes, showing positive associations for Aureobasidium pullulans and Alternaria sp. in productive areas, and positive associations for Fusarium sp. and Mortierella sp. were found in non‐productive soils. Finally, some significant OTUs were identified and associated to ascocarp producing patches, which could serve as predictive and location markers of desert truffle production

Desert truffle cultivation : New insights into mycorrhizal symbiosis, water-stress adaptation strategies and plantation management

Las trufas del desierto son hongos micorrícicos que desarrollan cuerpos fructíferos hipogeos y cuyo hábitat se limita a zonas áridas y semiáridas, principalmente de la cuenca del Mediterráneo y de Oriente Medio. De entre las trufas del desierto destacan los géneros Terfezia y Tirmania, cuyas especies forman ectendomicorrizas con plantas del género Helianthemum. La especie Terfezia claveryi Chatin fue la primera trufa del desierto en ser cultivada junto con la planta hospedante Helianthemum almeriense Pau. La presente tesis tiene el objetivo de aumentar nuestro conocimiento acerca de las trufas del desierto. Para cumplir este objetivo se propusieron cinco objetivos parciales cuyo desarrollo ha dado lugar a diversos resultados y conclusiones. Para evaluar el papel de catalasas fúngicas en la simbiosis T. claveryi y H. almeriense, se purificó una catalasa TcCAT-1 de ascocarpo de T. claveryi que fue caracterizada mediante técnicas bioquímicas, cromatográficas y bioinformáticas. Además, se midieron los niveles de transcripción del gen TcCAT-1 mediante PCR cuantitativa. Los resultados sugieren que TcCAT-1 puede jugar un papel importante durante la micorrización; además, este estudio ha puesto de manifiesto los beneficios de esta micorrización en la respuesta al estrés hídrico. Por otra parte, se secuenciaron los genomas de T. claveryi y Tirmania nivea y el transcriptoma de T. claveryi en micelio libre, micorriza bien regada y micorriza sometida a estrés hídrico; posteriormente se llevó a cabo el análisis bioinformático de todos esos datos. Se han descrito por primera vez los genes MAT de estas especies. También se estudió el patrón de expresión de las enzimas que actúan sobre la pared celular vegetal; este estudio ha revelado una regulación a la baja de estos genes durante la sequía, al mismo tiempo que se producía un cambio de tipo de micorrización, pasando de intercelular a intracelular. En general, las características descritas en este apartado están asociadas al tipo de micorriza formada por estas especies y a su adaptación a los hábitats secos. También se realizó un estudio de los datos que se han ido recogiendo durante 15 años de una plantación experimental de T. claveryi x H. almeriense con el objetivo de determinar los parámetros que más influyen en las fructificaciones de trufas del desierto y en qué momentos del año tienen una importancia mayor. Tras el análisis estadístico de los datos se puso de manifiesto que el índice de aridez en otoño y el potencial hídrico del suelo desde otoño a primavera, parecen ser los parámetros claves. Estos resultados nos han permitido proponer distintos modelos de manejo de plantaciones. En otro de los capítulos de esta tesis se caracterizaron las respuestas morfo-fisio-moleculares de la planta micorrizada H. almeriense durante primavera. Medidas de intercambio gaseoso y de cuantificación de la expresión de determinados genes de acuaporinas, pusieron de manifiesto un cambio brusco en la fenología de la planta que parece estar determinado, mayoritariamente, por el déficit de presión de vapor (DPV). Gracias a este resultado se ha determinado un valor umbral de DPV que está relacionado con la producción de trufas del desierto, y podemos concluir que, en líneas generales, cuanto más tarde se alcance este umbral en la primavera, mayores serán las producciones. Finalmente, con el fin de estudiar la viabilidad de este cultivo si se produjese un aumento en concentración de CO2 atmosférico, se realizó un ensayo en el que sometió a un grupo de H. almeriense micorrizadas con T. claveryi a una elevada concentración de CO2 combinado con un progresivo aumento de la sequía. Los resultados de este ensayo revelan que los efectos de una concentración elevada de CO2 pueden paliar, en parte, los efectos negativos del aumento de la sequía. Summary Desert truffles are mycorrhizal fungi that develop hypogeus fruiting bodies and inhabit arid and semiarid areas, such as the Mediterranean basin or the Middle East. Among desert truffles genera, Terfezia and Tirmania are of special importance. The species that belong to these genera mostly establish ectendomycorrhizal symbiosis with plants of Helianthemum genus. Terfezia claveryi Chatin was the first desert truffle cultivated, using Helianthemum almeriense Pau as its host. This thesis has the objective of deepen the knowledge of desert truffles and their cultivation. To achieve this goal, five specific objectives were proposed that have resulted in several results and conclusions. In order to evaluate the role of fungal catalases in T. claveryi x H. almeriense symbiosis, a catalase, TcCAT-1, from T. claveryi ascocarps was purified to homogeneity and characterized using biochemical, chromatographic and bioinformatic techniques. In addition, transcription levels of the gene TcCAT-1 were measured by quantitative PCR. The results obtained suggest an important role of TcCAT-1 in the mycorrhization; in addition, this study has revealed the benefits of this mycorrhization in the response to water-stress. The genomes of T. claveryi and Tirmania nivea and the T. claveryi transcriptome from free living mycelium, well-watered mycorrhiza and drought-stressed mycorrhiza were sequenced and analyzed with bioinformatics tools. MAT genes have been described in these species for the first time. Furthermore, the expression pattern of plant cell wall degrading enzymes revealed a generalized downregulation of these genes under water-stress conditions. This event occurs in parallel with the type of mycorrhiza that is formed, which changes from intercellular to intracellular. In summary, the genomic and transcriptomic features described in this thesis are associated with the type of mycorrhiza and the dry environments inhabited by these species. During this thesis, data collected during 15 years of an experimental orchard of T. claveryi x H. almeriense were analyzed in order to know what environmental parameters and at what times of the year determine the production of desert truffle the most. After the statistical analysis of the data, we found that autumn first, and spring, secondly, are the key moments that determine T. claveryi yield. Specifically, the aridity index in autumn and the soil water potential from autumn to spring are the most important parameters. With this in mind, several management models are proposed. The morpho-physio-molecular responses of the H. almeriense mycorrhizal plant were characterized during spring. Using gas-exchange and molecular techniques, an abrupt phenology switch was revealed. This switch seems to be determined primarily by the vapor pressure deficit (VPD). Thanks to this, a threshold value of VPD has been established that is related to the desert truffle fructifications: in general terms, the later this threshold is reached, the higher the desert truffle yield. In order to evaluate the viability of this crop under the foreseeable increase in atmospheric CO2 concentrations in the following years, an assay was carried out. This assay consisted of growing a group of T. claveryi mycorrhizal H. almeriense plants under high concentrations of atmospheric CO2, combined with a progressive increase in water-stress conditions. The results of this assay reveal that the effects of a high atmospheric concentration of CO2 partially counteract the negative effects derived from water-stress

Spring stomatal response to vapor pressure deficit as a marker for desert truffle fruiting

©. This manuscript version is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by /4.0/ This document is the Accepted version of a Published Work that appeared in final form in [Mycorrhiza]. To access the final edited and published work see[https://doi.org/10.1007/s00572-020-00966-8]The cultivation of desert truffle Terfezia claveryi using Helianthemum almeriense as a host plant has recently become a solid alternative crop in the Mediterranean region due to its adaptation to arid and semiarid ecosystems, which are expected to increase during the following years because of climate change. However, management models are still being developed in order to improve and stabilize the production, which varies greatly from one year to another. According to gatherers and farmers, one of the key factors for desert truffle production is the plant phenology in spring, which, in turn, depends on environmental conditions. In this manuscript, we have characterized the physiological, morphological, and molecular responses of the mycorrhizal plants in spring, coinciding with the fructification period of the plant and fungal species. Thanks to this characterization, a sigmoidal relationship between stomatal conductance and vapor pressure deficit (VPD)was found,which can be used as amarker of plant phenological switch. In order to confirmthat this phenology status is related to desert truffle fructification, this marker has been successfully correlated to total truffle production. The results of this manuscript suppose a big step forward that will help to develop management models for the desert truffle crop

The crop of desert truffle depends on agroclimatic parameters during two key annual periods

Desert truffles have become an alternative agricultural crop in semiarid areas of the Iberian Peninsula due to their much appreciated edible value and their low water requirements for cultivation. Although most studies related to desert truffle production point to the sole importance of precipitation, this work is the first systematic study carried out to characterize whether other important agroclimatic parameters, for example reference evapotranspiration, soil water potential, relative air humidity %, aridity index or air vapour pressure deficit, may have an impact on a desert truffle production in an orchard with mycorrhizal plants of Helianthemum almeriense × Terfezia claveryi for 15 years from the plantation. The results show for the first time that T. claveryi production has two key periods, during its annual cycle: autumn (September to October) and spring (end of March). The aridity index and soil water potential seem to be the most manageable parameters in the field and can be easily controlled by applying irrigation during the abovementioned periods. Agroclimatic parameters can influence the final crop a long time before the desert truffle fruiting season contrary to what happens with other edible mycorrhizal mushrooms. Four different models to manage desert truffle plantations are proposed based on these agroclimatic parameters in order to optimize and stabilize carpophore fructifications over the years

Purification and characterization of Terfezia claveryi TcCAT-1, a desert truffle catalase upregulated in mycorrhizal symbiosis.

Terfezia claveryi Chatin is a mycorrhizal fungus that forms ectendomycorrhizal associations with plants of Helianthemum genus. Its appreciated edibility and drought resistance make this fungus a potential alternative crop in arid and semiarid areas of the Mediterranean region. In order to increase the knowledge about the biology of this fungus in terms of mycorrhiza formation and response to drought stress, a catalase from T. claveryi (TcCAT-1) has been purified to apparent homogeneity and biochemically characterized; in addition, the expression pattern of this enzyme during different stages of T. claveryi biological cycle and under drought stress conditions are reported. The results obtained, together with the phylogenetic analysis and homology modeling, indicate that TcCAT-1 is a homotetramer large subunit size monofunctional-heme catalase belonging to Clade 2. The highest expression of this enzyme occurs in mature mycorrhiza, revealing a possible role in mycorrhiza colonization, but it is not upregulated under drought stress. However, the H2O2 content of mycorrhizal plants submitted to drought stress is lower than in well watered treatments, suggesting that mycorrhization improves the plant's oxidative stress response, although not via TcCAT-1 upregulation

Elevated atmospheric CO 2 modifies responses to water-stress and flowering of Mediterranean desert truffle mycorrhizal shrubs

©. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Accepted version of a Published Work that appeared in final form in [Physiologia Plantarum]. To access the final edited and published work see[10.1111/ppl.13190]Predicted increases in atmospheric concentration of carbon dioxide (CO2) coupled with increased temperatures and drought are expected to strongly influence the development of most of the plant species in the world, espe- cially in areas with high risk of desertification like the Mediterranean basin. Helianthemum almeriense is an ecologically important Mediterranean shrub with an added interest because it serves as the host for the Terfezia cla- veryi mycorrhizal fungus, which is a desert truffle with increasingly commer- cial interest. Although both plant and fungi are known to be well adapted to dry conditions, it is still uncertain how the increase in atmospheric CO2 will influence them. In this article we have addressed the physiological responses of H. almeriense × T. claveryi mycorrhizal plants to increases in atmospheric CO2 coupled with drought and high vapor pressure deficit. This work reports one of the few estimations of mesophyll conductance in a drought deciduous Mediterranean shrub and evaluates its role in photosyn- thesis limitation. High atmospheric CO2 concentrations help desert truffle mycorrhizal plants to cope with the adverse effects of progressive drought during Mediterranean springs by improving carbon net assimilation, intrinsic water use efficiency and dispersal of the species through increased flowering event

Elevated atmospheric CO 2

Predicted increases in atmospheric concentration of carbon dioxide (CO2) coupled with increased temperatures and drought are expected to strongly influence the development of most of the plant species in the world, especially in areas with high risk of desertification like the Mediterranean basin. Helianthemum almeriense is an ecologically important Mediterranean shrub with an added interest because it serves as the host for the Terfezia claveryi mycorrhizal fungus, which is a desert truffle with increasingly commercial interest. Although both plant and fungi are known to be well adapted to dry conditions, it is still uncertain how the increase in atmospheric CO2 will influence them. In this article we have addressed the physiological responses of H. almeriense × T. claveryi mycorrhizal plants to increases in atmospheric CO2 coupled with drought and high vapor pressure deficit. This work reports one of the few estimations of mesophyll conductance in a drought deciduous Mediterranean shrub and evaluates its role in photosynthesis limitation. High atmospheric CO2 concentrations help desert truffle mycorrhizal plants to cope with the adverse effects of progressive drought during Mediterranean springs by improving carbon net assimilation, intrinsic water use efficiency and dispersal of the species through increased flowering events

Desert truffle genomes reveal their reproductive modes and new insights into plant-fungal interaction and ectendomycorrhizal lifestyle.

Desert truffles are edible hypogeous fungi forming ectendomycorrhizal symbiosis with plants of Cistaceae family. Knowledge about the reproductive modes of these fungi and the molecular mechanisms driving the ectendomycorrhizal interaction is lacking. Genomes of the highly appreciated edible desert truffles Terfezia claveryi Chatin and Tirmania nivea Trappe have been sequenced and compared with other Pezizomycetes. Transcriptomes of T.&nbsp;claveryi&nbsp;×&nbsp;Helianthemum almeriense mycorrhiza from well-watered and drought-stressed plants, when intracellular colonizations is promoted, were investigated. We have identified the fungal genes related to sexual reproduction in desert truffles and desert-truffles-specific genomic and secretomic features with respect to other Pezizomycetes, such as the expansion of a large set of gene families with unknown Pfam domains and a number of species or desert-truffle-specific small secreted proteins differentially regulated in symbiosis. A core set of plant genes, including carbohydrate, lipid-metabolism, and defence-related genes, differentially expressed in mycorrhiza under both conditions was found. Our results highlight the singularities of desert truffles with respect to other mycorrhizal fungi while providing a first glimpse on plant and fungal determinants involved in ecto to endo symbiotic switch that occurs in desert truffle under dry conditions

Desert truffle genomes reveal their reproductive modes and new insights into plant-fungal interaction and ectendomycorrhizal lifestyle.

Desert truffles are edible hypogeous fungi forming ectendomycorrhizal symbiosis with plants of Cistaceae family. Knowledge about the reproductive modes of these fungi and the molecular mechanisms driving the ectendomycorrhizal interaction is lacking. Genomes of the highly appreciated edible desert truffles Terfezia claveryi Chatin and Tirmania nivea Trappe have been sequenced and compared with other Pezizomycetes. Transcriptomes of T.&nbsp;claveryi&nbsp;×&nbsp;Helianthemum almeriense mycorrhiza from well-watered and drought-stressed plants, when intracellular colonizations is promoted, were investigated. We have identified the fungal genes related to sexual reproduction in desert truffles and desert-truffles-specific genomic and secretomic features with respect to other Pezizomycetes, such as the expansion of a large set of gene families with unknown Pfam domains and a number of species or desert-truffle-specific small secreted proteins differentially regulated in symbiosis. A core set of plant genes, including carbohydrate, lipid-metabolism, and defence-related genes, differentially expressed in mycorrhiza under both conditions was found. Our results highlight the singularities of desert truffles with respect to other mycorrhizal fungi while providing a first glimpse on plant and fungal determinants involved in ecto to endo symbiotic switch that occurs in desert truffle under dry conditions