Kearney Agricultural Research and Extension Center

Drought stress is a major obstacle to crop productivity, and the severity and frequency of drought are expected to increase in the coming century. Certain root-associated bacteria have been shown to mitigate the negative effects of drought stress on plant growth, and manipulation of the crop microbiome is an emerging strategy for overcoming drought stress in agricultural systems, yet the effect of drought on the development of the root microbiome is poorly understood. Through 16S rRNA amplicon and metatranscriptome sequencing, as well as root metabolomics, we demonstrate that drought delays the development of the early sorghum root microbiome and causes increased abundance and activity of monoderm bacteria, which lack an outer cell membrane and contain thick cell walls. Our data suggest that altered plant metabolism and increased activity of bacterial ATP-binding cassette (ABC) transporter genes are correlated with these shifts in community composition. Finally, inoculation experiments with monoderm isolates indicate that increased colonization of the root during drought can positively impact plant growth. Collectively, these results demonstrate the role that drought plays in restructuring the root microbiome and highlight the importance of temporal sampling when studying plant-associated microbiomes.

Plant response to drought stress involves fungi and bacteria that live on and in plants and in the rhizosphere, yet the stability of these myco- and micro-biomes remains poorly understood. We investigate the resistance and resilience of fungi and bacteria to drought in an agricultural system using both community composition and microbial associations. Here we show that tests of the fundamental hypotheses that fungi, as compared to bacteria, are (i) more resistant to drought stress but (ii) less resilient when rewetting relieves the stress, found robust support at the level of community composition. Results were more complex using all-correlations and co-occurrence networks. In general, drought disrupts microbial networks based on significant positive correlations among bacteria, among fungi, and between bacteria and fungi. Surprisingly, co-occurrence networks among functional guilds of rhizosphere fungi and leaf bacteria were strengthened by drought, and the same was seen for networks involving arbuscular mycorrhizal fungi in the rhizosphere. We also found support for the stress gradient hypothesis because drought increased the relative frequency of positive correlations.

Community assembly of crop-associated fungi is thought to be strongly influenced by deterministic selection exerted by the plant host, rather than stochastic processes. Here we use a simple, sorghum system with abundant sampling to show that stochastic forces (drift or stochastic dispersal) act on fungal community assembly in leaves and roots early in host development and when sorghum is drought stressed, conditions when mycobiomes are small. Unexpectedly, we find no signal for stochasticity when drought stress is relieved, likely due to renewed selection by the host. In our experimental system, the host compartment exerts the strongest effects on mycobiome assembly, followed by the timing of plant development and lastly by plant genotype. Using a dissimilarity-overlap approach, we find a universality in the forces of community assembly of the mycobiomes of the different sorghum compartments and in functional guilds of fungi.

California is a global leader in the agricultural sector and produces more than 400 types of commodities. The state produces over a third of the country’s vegetables and two-thirds of its fruits and nuts. Despite being highly productive, current and future climate change poses many challenges to the agricultural sector. This paper provides a summary of the current state of knowledge on historical and future trends in climate and their impacts on California agriculture. We present a synthesis of climate change impacts on California agriculture in the context of: (1) historic trends and projected changes in temperature, precipitation, snowpack, heat waves, drought, and flood events; and (2) consequent impacts on crop yields, chill hours, pests and diseases, and agricultural vulnerability to climate risks. Finally, we highlight important findings and directions for future research and implementation. The detailed review presented in this paper provides sufficient evidence that the climate in California has changed significantly and is expected to continue changing in the future, and justifies the urgency and importance of enhancing the adaptive capacity of agriculture and reducing vulnerability to climate change. Since agriculture in California is very diverse and each crop responds to climate differently, climate adaptation research should be locally focused along with effective stakeholder engagement and systematic outreach efforts for effective adoption and implementation. The expected readership of this paper includes local stakeholders, researchers, state and national agencies, and international communities interested in learning about climate change and California’s agriculture.

Recent studies have demonstrated that drought leads to dramatic, highly conserved shifts in the root microbiome. At present, the molecular mechanisms underlying these responses remain largely uncharacterized. Here we employ genome-resolved metagenomics and comparative genomics to demonstrate that carbohydrate and secondary metabolite transport functionalities are overrepresented within drought-enriched taxa. These data also reveal that bacterial iron transport and metabolism functionality is highly correlated with drought enrichment. Using time-series root RNA-Seq data, we demonstrate that iron homeostasis within the root is impacted by drought stress, and that loss of a plant phytosiderophore iron transporter impacts microbial community composition, leading to significant increases in the drought-enriched lineage, Actinobacteria. Finally, we show that exogenous application of iron disrupts the drought-induced enrichment of Actinobacteria, as well as their improvement in host phenotype during drought stress. Collectively, our findings implicate iron metabolism in the root microbiome's response to drought and may inform efforts to improve plant drought tolerance to increase food security.

(L.) Moench] is a critical food, forage, and emerging bioenergy crop that is notably drought-tolerant. We conducted a large-scale field experiment, imposing preflowering and postflowering drought stress on 2 genotypes of sorghum across a tightly resolved time series, from plant emergence to postanthesis, resulting in a dataset of nearly 400 transcriptomes. We observed a fast and global transcriptomic response in leaf and root tissues with clear temporal patterns, including modulation of well-known drought pathways. We also identified genotypic differences in core photosynthesis and reactive oxygen species scavenging pathways, highlighting possible mechanisms of drought tolerance and of the delayed senescence, characteristic of the stay-green phenotype. Finally, we discovered a large-scale depletion in the expression of genes critical to arbuscular mycorrhizal (AM) symbiosis, with a corresponding drop in AM fungal mass in the plants' roots.

Species of family Botryosphaeriaceae and genus Diaporthe (anamorph: genus Phomopsis, family Diaporthaceae) were reported and caused diseases on various fruit and nut trees in California. In the last several years, diseases on English walnut (Juglans regia) caused by species of Botryosphaeriaceae and Diaporthe were observed frequently in California. Disease symptoms include stem canker; shoot canker and blight; twig, leaf, and fruit blight; and necrotic leaf lesions. Isolates of the pathogen were collected from English walnut in 13 counties in California. The aims of this study were to identify these isolates and to test their pathogenicity to English walnut cultivars. In total, 159 California isolates were identified based on comparisons of DNA sequence data of the internal transcribed spacer, translation elongation factor 1-α, and β-tubulin gene regions, and combined with the morphological features of the cultures and conidia. Research results revealed that isolates represent 10 species of Botryosphaeriaceae and two species of Diaporthe. These species include Botryosphaeria dothidea, Diplodia mutila, D. seriata, Dothiorella iberica, Lasiodiplodia citricola, Neofusicoccum mediterraneum, N. nonquaesitum, N. parvum, N. vitifusiforme, Neoscytalidium dimidiatum, Diaporthe neotheicola, and D. rhusicola. Pathogenicity on three English walnut cultivars ('Chandler', 'Tulare', and 'Vina') using a mycelium plug inoculation method revealed that all these species are pathogenic to all the tested cultivars, with L. citricola and N. parvum being the most pathogenic species, followed by N. mediterraneum, N. dimidiatum, and B. dothidea. Chandler was more tolerant to infection than Tulare and Vina. Results in this study determined that multiple numbers of the Botryosphaeriaceae fungi and two Diaporthe spp. cause cankers and blights of English walnut and vary in their virulence from highly to slightly virulent, respectively.

The ecology of fungi lags behind that of plants and animals because most fungi are microscopic and hidden in their substrates. Here, we address the basic ecological process of fungal succession in nature using the microscopic, arbuscular mycorrhizal fungi (AMF) that form essential mutualisms with 70-90% of plants. We find a signal for temporal change in AMF community similarity that is 40-fold stronger than seen in the most recent studies, likely due to weekly samplings of roots, rhizosphere and soil throughout the 17 weeks from seedling to fruit maturity and the use of the fungal DNA barcode to recognize species in a simple, agricultural environment. We demonstrate the patterns of nestedness and turnover and the microbial equivalents of the processes of immigration and extinction, that is, appearance and disappearance. We also provide the first evidence that AMF species co-exist rather than simply co-occur by demonstrating negative, density-dependent population growth for multiple species. Our study shows the advantages of using fungi to test basic ecological hypotheses (e.g., nestedness v. turnover, immigration v. extinction, and coexistence theory) over periods as short as one season.

Assessment of the nitrogen status of grapevines with high spatial, temporal resolution offers benefits in fertilizer use efficiency, crop yield and quality, and vineyard uniformity. The primary objective of this study was to develop a robust predictive model for grapevine nitrogen estimation at bloom stage using high-resolution multispectral images captured by an unmanned aerial vehicle (UAV). Aerial imagery and leaf tissue sampling were conducted from 150 grapevines subjected to five rates of nitrogen applications. Subsequent to appropriate pre-processing steps, pixels representing the canopy were segmented from the background per each vine. First, we defined a binary classification problem using pixels of three vines with the minimum (low-N class) and two vines with the maximum (high-N class) nitrogen concentration. Following optimized hyperparameters configuration, we trained five machine learning classifiers, including support vector machine (SVM), random forest, XGBoost, quadratic discriminant analysis (QDA), and deep neural network (DNN) with fully-connected layers. Among the classifiers, SVM offered the highest F1-score (82.24%) on the test dataset at the cost of a very long training time compared to the other classifiers. Alternatively, QDA and XGBoost required the minimum training time with promising F1-score of 80.85% and 80.27%, respectively. Second, we transformed the classification into a regression problem by averaging the posterior probability of high-N class for all pixels within each of 150 vines. XGBoost exhibited a slightly larger coefficient of determination (R2 = 0.56) and lower root mean square error (RMSE) (0.23%) compared to other learning methods in the prediction of nitrogen concentration of all vines. The proposed approach provides values in (i) leveraging high-resolution imagery, (ii) investigating spatial distribution of nitrogen across a vine’s canopy, and (iii) defining spatial zones for nitrogen application and smart sampling.

Aflatoxin contamination of important food and feed crops occurs frequently in warm tropical and subtropical regions. The contamination is caused mainly by Aspergillus flavus and A. parasiticus. Aflatoxin contamination negatively affects health and trade sectors and causes economic losses to agricultural industries. Many pre- and post-harvest technologies can limit aflatoxin contamination but may not always reduce aflatoxin concentrations below tolerance thresholds. However, the use of atoxigenic (non-toxin producing) isolates of A. flavus to competitively displace aflatoxin producers is a practical strategy that effectively limits aflatoxin contamination in crops from field to plate. Biocontrol products formulated with atoxigenic isolates as active ingredients have been registered for use in the US, several African nations, and one such product is in final stages of registration in Italy. Many other nations are seeking to develop biocontrol products to protect their crops. In this review article we present an overview of the biocontrol technology, explain the basis to select atoxigenic isolates as active ingredients, describe how formulations are developed and tested, and describe how a biocontrol product is used commercially. Future perspectives on formulations of aflatoxin biocontrol products, along with other important topics related to the aflatoxin biocontrol technology are also discussed.

Botryosphaeria panicle and shoot blight was considered as one of the single greatest threats to the California pistachio industry in the last two decades. A large number of fungi with typical morphological characteristics of Botryosphaeriaceae and Diaporthe were collected from pistachios in 18 counties in California and deposited in our culture collection. The aims of this study were to identify these isolates, recognize the distribution of these fungal species and test their pathogenicity to pistachio cultivars. A total of 304 California isolates were identified based on comparisons of DNA sequence data of the ITS, TEF-1α and β-tubulin gene regions, and combined with the morphological features of the cultures and conidia. Research results showed that California isolates represent eight species of Botryosphaeriaceae and one species of Diaporthe. These species include Botryosphaeria dothidea, Diplodia seriata, Dothiorella iberica, Dot. sarmentorum, Lasiodiplodia citricola, L. gilanensis, Neofusicoccum mediterraneum, Neof. vitifusiforme and Diaporthe chamaeropis. Of the Botryosphaeriaceae, 86 % of the isolates were identified as Neof. mediterraneum, which distributed in all the sampled counties. On pistachio trees, in addition to isolates from California, Neof. mediterraneum from Arizona, Neof. australe from Australia, B. dothidea, Neof. parvum and Dia. viticola from Greece were also identified. Pathogenicity of the California fungi on pistachio cultivars, Kerman (female) and Peters (male), using a mycelium plug and conidial suspension inoculation methods showed that all these species are pathogenic to the two tested pistachio cultivars, with L. citricola, L. gilanensis being the most pathogenic species, followed by Neof. mediterraneum. This study represents the first comprehensive work on the species identification, distribution and pathogenicity of Botryosphaeriaceae and Diaporthe on pistachio in California.

The atoxigenic strain Aspergillus flavus AF36, which has been extensively used as a biocontrol agent in commercial corn and cotton fields to reduce aflatoxin contamination, was applied in research pistachio orchards from 2002 to 2005 and in commercial pistachio orchards from 2008 to 2011. AF36 was applied as hyphae-colonized steam-sterilized wheat seed (the same product and same application rate as used in cotton fields). In all orchards, applying the wheat-AF36 product substantially increased the proportion of vegetative compatibility group (VCG) YV36, the VCG to which AF36 belongs, within A. flavus soil communities. Application of the AF36 product in additional years further increased YV36 in the soil until it composed 93% of the A. flavus isolates in treated commercial orchards. Nonetheless, application of the AF36 product did not result in increased incidence of kernel decay of the nuts. For nuts harvested from commercial orchards, reductions in percentages of samples contaminated with aflatoxin from treated orchards (relative to that for untreated orchards) ranged from 20 to 45%, depending on the year. Because of the high value of pistachio nuts and the costs associated with rejection of shipments due to aflatoxin contamination, these reductions are significant and valuable to the pistachio industry.

Over two consecutive seasons, 16 olive orchards with trees exhibiting dieback symptoms on branches were surveyed in southern Spain. The six dominant fungal species recovered were characterized by means of phenotypic observations, DNA analysis (by sequencing of the internal transcribed spacer, β-tubulin, and large subunit nuclear ribosomal DNA regions), and pathogenicity tests. Additionally, three isolates collected from Tunisian olive trees showing similar dieback symptoms, one isolate of Colletotrichum godetiae, and a reference isolates of Neofusicoccum mediterraneum were included. The resistance of the 11 most important table cultivars to N. mediterraneum and Botryosphaeria dothidea, the causal agent of "escudete" (small shield) of fruit, was studied by the inoculation of branches and immature fruit, respectively. The species Cytospora pruinosa, N. mediterraneum, Nothophoma quercina, Comoclathris incompta, and Diaporthe sp. were identified. Only N. mediterraneum and C. incompta were able to induce the typical dieback symptoms and cankers that affected the development of the plants. The species N. mediterraneum was the most virulent among the evaluated species, although differences in virulence among its isolates were observed. The remaining fungal species were weakly pathogenic to nonpathogenic on plants. According to resistance tests, 'Gordal Sevillana' and 'Manzanilla Cacereña' were the most susceptible to branch dieback caused by N. mediterraneum. Furthermore, the fruit of 'Aloreña de Atarfe' and 'Manzanilla de Sevilla' were the most susceptible to B. dothidea. Knowledge of the etiology and cultivar resistance of these diseases will help to establish better control measures.

Gray mold caused by Botrytis cinerea is a major postharvest disease of blueberry grown in the Central Valley of California and western Washington State. Sensitivities to boscalid, cyprodinil, fenhexamid, fludioxonil, and pyraclostrobin, representing five different fungicide classes, were examined for 249 (California) and 106 (Washington) B. cinerea isolates recovered from decayed blueberry fruit or flowers. In California and Washington, 7 and 17 fungicide-resistant phenotypes, respectively, were detected: 66 and 49% of the isolates were resistant to boscalid, 20 and 29% were moderately resistant to cyprodinil, 29 and 29% were resistant to fenhexamid, and 66 and 55% were resistant to pyraclostrobin. All isolates from California were sensitive to fludioxonil, whereas 70% of the isolates from Washington showed reduced sensitivity to fludioxonil. In California, 26 and 30% of the isolates were resistant to two and three classes of fungicides, respectively. In Washington, 31, 14, 16, and 9% of the isolates were resistant to two, three, four, and five classes of fungicides, respectively. Inherent risk of the development of resistance to quinone outside inhibitor (QoI) fungicides was assessed by detecting the presence of the Bcbi-143/144 intron in gene cytb. The intron was detected in 11.8 and 40% of the isolates in California and Washington, respectively, suggesting that the risk of QoI resistance is higher in California than in Washington. On detached blueberry fruit inoculated with 11 isolates exhibiting different fungicide-resistant phenotypes, most fungicides failed to control gray mold on fruit inoculated with the respective resistant phenotypes but the mixture of cyprodinil and fludioxonil was effective against all fungicide-resistant phenotypes tested. Our findings would be useful in designing and implementing fungicide resistance management spray programs for control of gray mold in blueberry.

Resistance of Alternaria alternata to boscalid, the first succinate dehydrogenase inhibitor (SDHI) fungicide labeled on pistachio, has become a common occurrence in California pistachio orchards and affects the performance of this fungicide. In this study, we established the baseline sensitivities of A. alternata to the new SDHIs fluopyram, fluxapyroxad, and penthiopyrad and assessed their cross resistance patterns with boscalid. Examination of the effective fungicide concentration that inhibits mycelial growth to 50% relative to the control (EC 50 ) for 50 baseline isolates revealed that the majority were sensitive to boscalid, penthiopyrad, fluopyram, and fluxapyroxad. Analysis of EC 50 values for boscalid for 117 A. alternata isolates originating from boscalid-exposed orchards showed that 44, 3, 1, and 69 isolates had sensitive, reduced sensitivity, moderately resistant, and highly resistant boscalid phenotypes, respectively. Molecular investigation of the occurrence of known SDH mutations showed that, among the 69 isolates highly resistant to boscalid, 44, 2, 14, and 1 isolates possessed the mutations leading to the H277Y, H277R, H134R, and H133R amino acid substitutions in AaSDHB, AaSDHB, AaSDHC, and AaSDHD subunits, respectively. Some SDHB or SDHC mutants displayed highly sensitive, sensitive, or reduced sensitivity phenotypes toward penthiopyrad or fluxapyroxad, whereas other had low, moderate, or high levels of resistance to these fungicides. In contrast, all the SDHB mutants were sensitive to fluopyram, while 10, 5, and 1 SDHC mutants had sensitive, reduced sensitivity, and moderately resistant fluopyram phenotypes, respectively. The SDHD mutant had reduced sensitivity to fluopyram and penthiopyrad but was highly resistant to fluxapyroxad. The discrepancies of cross-resistance patterns between SDHIs suggest that their binding sites in complex II may differ slightly and that additional mechanisms of resistance to these compounds are likely involved. Ultimately, the findings of this study should lead to the rational and sustained deployment of new SDHIs in Alternaria late blight spray programs.

Sorghum bicolor (L.) Moench, the fifth most important cereal worldwide, is a multi-use crop for feed, food, forage and fuel. To enhance the sorghum and other important crop plants, establishing gene function is essential for their improvement. For sorghum, identifying genes associated with its notable abiotic stress tolerances requires a detailed molecular understanding of the genes associated with those traits. The limits of this knowledge became evident from our earlier in-depth sorghum transcriptome study showing that over 40% of its transcriptome had not been annotated. Here, we describe a full spectrum of tools to engineer, edit, annotate and characterize sorghum's genes. Efforts to develop those tools began with a morphogene-assisted transformation (MAT) method that led to accelerated transformation times, nearly half the time required with classical callus-based, non-MAT approaches. These efforts also led to expanded numbers of amenable genotypes, including several not previously transformed or historically recalcitrant. Another transformation advance, termed altruistic, involved introducing a gene of interest in a separate Agrobacterium strain from the one with morphogenes, leading to plants with the gene of interest but without morphogenes. The MAT approach was also successfully used to edit a target exemplary gene, phytoene desaturase. To identify single-copy transformed plants, we adapted a high-throughput technique and also developed a novel method to determine transgene independent integration. These efforts led to an efficient method to determine gene function, expediting research in numerous genotypes of this widely grown, multi-use crop.

The Botrytis cinerea species complex comprises two cryptic species, originally referred to Group I and Group II based on Bc-hch gene RFLP haplotyping. Group I was described as a new cryptic species B. pseudocinerea During a survey of Botrytis spp. causing gray mold in blueberries and table grapes in the Central Valley of California, six isolates, three from blueberries and three from table grapes, were placed in Group I but had a distinct morphological character with conidiophores significantly longer than those of B. cinerea and B. pseudocinerea We compared these with B. cinerea and B. pseudocinerea by examining morphological and physiological characters, sensitivity to fenhexamid and phylogenetic analysis inferred from sequences of three nuclear genes. Phylogenetic analysis with the three partial gene sequences encoding glyceraldehyde-3-phosate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60) and DNA-dependent RNA polymerase subunit II (RPB2) supported the proposal of a new Botrytis species, B. californica, which is closely related genetically to B. cinerea, B. pseudocinerea and B. sinoviticola, all known as causal agents of gray mold of grapes. Botrytis californica caused decay on blueberry and table grape fruit inoculated with the fungus. This study suggests that B. californica is a cryptic species sympatric with B. cinerea on blueberries and table grapes in California.

Almond trees with trunk and branch cankers were observed in several orchards across almond-producing counties in California. Symptoms of cankers included bark lesions, discoloration of xylem tissues, longitudinal wood necrosis, and extensive gumming. Spur and shoot blight associated with rotted fruit were detected in two orchards in Kern County. The fungus Neoscytalidium dimidiatum was consistently recovered from the various cankers, infected fruit, and blighted shoots and its identity was confirmed based on phylogenetic and morphological studies. Phylogenetic analyses of the internal transcribed spacer, translation elongation factor 1-α, and β-tubulin genes comparing 47 strains from California with reference specimens within the family Botryosphaeriaceae and coupled with detailed morphological observations validated the identity of the pathogenic fungus. Pathogenicity tests conducted in the field using 1- to 2-year-old branches inoculated with mycelium plugs or conidial suspensions and attached fruit inoculated with conidial suspensions fulfilled Koch's postulates. N. dimidiatum appeared highly virulent in almond-producing cankers of up to 22 cm in length within 4 weeks using mycelium plug inoculations as well as severe fruit rot combined with spur blight on the fruit-bearing spurs. This study reports, for the first time, the fungus N. dimidiatum as a pathogen of almond in California causing canker, shoot blight, and fruit rot. Disease symptoms are described and illustrated.

The use of ensiled biomass sorghum enables implementation of relatively mild pretreatment conditions compared to non-ensiled sorghum and results in higher sugar yields, which reduces the biofuel production cost and associated carbon footprint.

Almond canker diseases are destructive and can reduce the yield as well as the lifespan of almond orchards. These diseases may affect the trunk and branches of both young and mature trees and can result in tree death soon after orchard establishment in severe cases. Between 2015 and 2018, 70 almond orchards were visited throughout the Central Valley of California upon requests from farm advisors for canker disease diagnosis. Two major canker diseases were identified, including Botryosphaeriaceae cankers and Ceratocystis canker. In addition, five less prevalent canker diseases were identified, including Cytospora, Eutypa, Diaporthe, Collophorina, and Pallidophorina canker. Seventy-four fungal isolates were selected for multilocus phylogenetic analyses of internal transcribed spacer region ITS1-5.8S-ITS2 and part of the translation elongation factor 1-α, β-tubulin, and glyceraldehyde 3-phosphate dehydrogenase gene sequences; 27 species were identified, including 12 Botryosphaeriaceae species, Ceratocystis destructans, five Cytospora species, Collophorina hispanica, four Diaporthe species, two Diatrype species, Eutypa lata, and Pallidophorina paarla. The most frequently isolated species were Ceratocystis destructans, Neoscytalidium dimidiatum, and Cytospora californica. Pathogenicity experiments on almond cultivar Nonpareil revealed that Neofusicoccum parvum, Neofusicoccum arbuti, and Neofusicoccum mediterraneum were the most virulent. Botryosphaeriaceae cankers were predominantly found in young orchards and symptoms were most prevalent on the trunks of trees. Ceratocystis canker was most commonly found in mature orchards and associated with symptoms found on trunks or large scaffold branches. This study provides a thorough examination of the diversity and pathogenicity of fungal pathogens associated with branch and trunk cankers of almond in California.