Origin of agricultural plant pathogens: Diversity and pathogenicity of Rhizoctonia fungi associated with native prairie grasses in the Sandhills of Nebraska

The Sandhills of Nebraska is a complex ecosystem, covering 50,000 km2 in central and western Nebraska and predominantly of virgin grassland. Grasslands are the most widespread vegetation in the U.S. and once dominated regions are currently cultivated croplands, so it stands to reason that some of the current plant pathogens of cultivated crops originated from grasslands, particularly soilborne plant pathogens. The anamorphic genus Rhizoctonia includes genetically diverse organisms that are known to be necrotrophic fungal pathogens, saprophytes, mycorrhiza of orchids, and biocontrol agents. This study aimed to evaluate the diversity of Rhizoctonia spp. on four native grasses in the Sandhills of Nebraska and determine pathogenicity to native grasses and soybean. In 2016 and 2017, a total of 84 samples were collected from 11 sites in the Sandhills, located in eight counties of Nebraska. The samples included soil and symptomatic roots from the four dominant native grasses: sand bluestem, little bluestem, prairie sandreed, and needle-and-thread. Obtained were 17 Rhizoctonia-like isolates identified, including five isolates of binucleate Rhizoctonia AG-F; two isolates each from binucleate Rhizoctonia AG-B, AG-C, and AG-K, Rhizoctonia solani AGs: AG-3, and AG-4; one isolate of binucleate Rhizoctonia AG-L, and one isolate of R. zeae. Disease severity was assessed for representative isolates of each AG in a greenhouse assay using sand bluestem, needle-and-thread, and soybean; prairie sandreed and little bluestem were unable to germinate under artificial conditions. On native grasses, all but two isolates were either mildly aggressive (causing 5–21% disease severity) or aggressive (21–35% disease severity). Among those, three isolates were cross-pathogenic on soybean, with R. solani AG-4 shown to be highly aggressive (86% disease severity). Thus, it is presumed that Rhizoctonia spp. are native to the sandhills grasslands and an emerging pathogen of crops cultivated may have survived in the soil and originate from grasslands.

Synergy Assessment for Plant Growth by Independent Joint Action Theory

Calculating the predicted biological efficacy of a mixture and determining the significance of the difference between the predicted efficacy and the measured efficacy of that mixture are fundamental when assessing the synergy of mixtures. The Independent Joint Action theory and Bliss’s formula are well-known and widely accepted for predicting pesticide mixture effects that are expressed in terms of percent mortality. Bliss’s formula, however, is not applicable to growth-affecting components, such as plant growth regulators. Therefore, there is an unmet need of critical importance: an appropriate method for assessing synergy of growth-affecting mixtures needs to be identified within the scientific community. The formula, G(1 + 2..n) = G1G2…Gn/(GCTL)n−1, which was derived from the Independent Joint Action theory, is presented for calculating the predicted efficacy for mixtures of growth-affecting components that either promote or inhibit growth. Its application is demonstrated by analyzing data from a greenhouse assay in which a mixture of S-abscisic acid and gibberellic acid was used to promote the growth of corn seedlings.

Soil Surface-Trapping of Tomato Leaf-Miner Flies Emerging from Underground Pupae with a Simple Electrostatic Cover of Seedbeds in a Greenhouse

In the present study, an electrostatic apparatus for trapping adult tomato leaf miner flies (Liriomyza sativae) emerging from underground pupae at the surface of a seedbed in an organic greenhouse was developed. The apparatus consisted of insulated iron rods arranged in parallel at set intervals and linked to a voltage generator, which supplied a negative charge to the rods, as well as non-insulated grounded iron rods with the same configuration. The two layers of insulated and non-insulated iron rods were arrayed in parallel to form a static electric field between the layers. The electric field created a strong attractive force capable of capturing flies that entered the field. In a greenhouse assay, the apparatus was placed horizontally above a seedbed in a greenhouse and surveyed for its ability to capture adult flies emerging from pupae that were introduced onto the seedbed beneath the apparatus. The results revealed that the apparatus effectively trapped all adult flies that emerged from the pupae and that it functioned stably while continuously operated during the entire period of the experiment. Thus, our novel apparatus is a promising tool for the physical control of adult tomato leaf miners in the insecticide-independent cultivation of greenhouse tomatoes.

Influence of Riparian Habitat and Ground Covers on Threecornered Alfalfa Hopper (Hemiptera: Membracidae) Populations in Vineyards

Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch disease, which affects wine grapes and leads to reduced crop yield and quality. While some virus spread can be attributed to the propagation of infected plant material, a greenhouse assay recently demonstrated that the threecornered alfalfa hopper (Membracidae: Spissistilus festinus Say) can transmit GRBV between grapevines. While S. festinus is not considered an economic pest of wine grapes, this species is present in California vineyards and their feeding can cause petiole girdling. Recent surveys have noted a correlation between S. festinus populations and GRBV-positive vines in vineyard areas adjacent to riparian habitat. Here, S. festinus populations were monitored over a 2-yr period at multiple vineyard sites adjacent to riparian habitats. At each site, insects were sampled from ground covers and the vine canopy at the vineyard edge and interior, and vines in both locations were evaluated for petiole girdling. Results indicate that there was no difference in abundance of S. festinus at the vineyard edge and interior. Populations in the vine canopy were highest in the late spring and early summer, and this was followed by the appearance of petiole girdling, indicating a key period of potential GRBV transmission. Furthermore, activity in the vine canopy appears to be amplified when the quality of ground covers is reduced as the season progresses. That said, overall populations of S. festinus were relatively low and additional work is needed to characterize the timing and efficiency of transmission under field conditions.

Bioprospection and molecular phylogeny of culturable endophytic fungi associated with yellow passion fruit

Endophytic microorganisms live inside the plants without causing any damage to their hosts. In the agricultural field, these endophytes might be a strategy of biological control for phytopathogens. We aimed to isolate endophytic fungi from yellow passion fruit (Passiflora edulis) leaves, evaluating its biocontrol capacity by in vitro antagonism against phytopathogen Colletotrichum sp. CNPU378. We also carried out greenhouse experiments in bean seedlings. A high colonization frequency was obtained (89%), and the molecular identification based on DNA sequencing attested Colletotrichum as the most frequent genus and minor occurrence of Curvularia endophytes. The endophytes tested showed different types of competitive interactions in in vitro antagonism inhibition rate ranging from 28.8 to 48.8%. There were 10 promising antagonists tested for their antagonist activity of crude extracts of secondary metabolites, in which strain PE-36 (20.8%) stood out among the other strains evaluated. In the greenhouse assay, plants inoculated only with endophyte Colletotrichum sp. PE-36 was symptomless and suggest that the endophyte strengthened the growth promotion in common bean plants, especially in the root length and number of leaves when compared to control plants and other treatments. Despite many fungi of Colletotrichum genus being described as causative agents of anthracnose, in this study, the plant sampled was colonized predominantly by Colletotrichum endophytes living in asymptomatic relationship. By the way, we come across a Colletotrichum sp. endophyte able to antagonize a Colletotrichum sp. pathogen.

Spinasterol, 22,23-Dihydrospinasterol and Fernenol from Citrullus Colocynthis L. with Aphicidal Activity against Cabbage Aphid Brevicoryne Brassicae L.

Brevicoryne brassicae is a problematic pest in cabbage and other field crops. Synthetic pesticides are used to control this pest, but they are injurious for human health and the environment. The present study aimed to purify and identify the active compounds from Citrullus colocynthis leaves with an appraisal of their efficacy against B. brassicae. Separation and purification were performed via different chromatographic techniques. Molecular analysis and chemical structures were recognized by mass spectrum (MS) and nuclear magnetic resonance (NMR), respectively. Moreover, in vitro and in vivo aphicidal activity was assessed using various concentrations, i.e., 6.25, 12.5, 25 and 50 µg/mL at 12, 24, 48 and 72 h exposure. The outcome shows that mass spectrum analyses of the purified compounds suggested the molecular formulae are C30H50O and C29H50O, C29H48O. The compounds were characterized as fernenol and a mixture of spinasterol, 22,23-dihydrospinasterol by 1H-NMR and 13C-NMR spectrum analysis. The toxicity results showed that the mixture of spinasterol and 22,23-dihydrospinasterol showed LC50 values of 32.36, 44.49 and 37.50 µg/mL by contact, residual and greenhouse assay at 72 h exposure, respectively. In contrast, fernenol recorded LC50 values as 47.99, 57.46 and 58.67 µg/mL, respectively. On the other hand, spinasterol, 22,23-dihydrospinasterol showed the highest mortality, i.e., 66.67%, 53.33% and 60% while, 30%, 23.33% and 25% mortality was recorded by fernenol after 72 h at 50 µg/mL by contact, residual and greenhouse assay, respectively. This study suggests that spinasterol, 22,23-dihydrospinasterol are more effective against B. brassicae which may be introduced as an effective and suitable substitute of synthetic chemical pesticides.

Genetic variation associated with PPO-inhibiting herbicide tolerance in sorghum

Herbicide application is crucial for weed management in most crop production systems, but for sorghum herbicide options are limited. Sorghum is sensitive to residual protoporphyrinogen oxidase (PPO)- inhibiting herbicides, such as fomesafen, and a long re-entry period is required before sorghum can be planted after its application. Improving sorghum for tolerance to such residual herbicides would allow for increased sorghum production and the expansion of herbicide options for growers. To investigate the underlying mechanism of tolerance to residual fomesafen, a genome-wide association mapping study was conducted using the sorghum biomass panel (SBP) and field-collected data, and a greenhouse assay was developed to confirm the field phenotypes. A total of 26 significant SNPs (FDR<0.05), spanning a 215.3 kb region, were detected on chromosome 3. The ten most significant SNPs included two in genic regions (Sobic.003G136800, and Sobic.003G136900) and eight SNPs in the intergenic region encompassing the genes Sobic.003G136700, Sobic.003G136800, Sobic.003G137000, Sobic.003G136900, and Sobic.003G137100. The gene Sobic.003G137100 (PPXI), which encodes the PPO1 enzyme, one of the targets of PPO-inhibiting herbicides, was located 12kb downstream of the significant SNP S03_13152838. We found that PPXI is highly conserved in sorghum and expression does not significantly differ between tolerant and sensitive sorghum lines. Our results suggest that PPXI most likely does not underlie the observed herbicide tolerance. Instead, the mechanism underlying herbicide tolerance in the SBP is likely metabolism-based resistance, possibly regulated by the action of multiple genes. Further research is necessary to confirm candidate genes and their functions.

SELEKSI BERBANTUKAN MARKA MOLEKULER UNTUK PEMBENTUKAN CALON VARIETAS PADI TURUNAN ESENSIAL SITU PATENGGANG

The utilization of molecular marker technology for Blast (Pyricularia grisea) resistance in rice breeding could accelerate and improve the precision of selection. This study aimed to identify and to select the BC3F2 progenies from Situ Patenggang and the opted Blast Monogenic Lines based on their resistance to blast disease in green house and field using molecular characterization. A total of 200-300 BC3F2 population strains resulted in 4 crosses between Situ Patenggang varieties and Monogenic Varieties IRBLta2-Re, IRBLkp-k60, IRBLi-F5 and IRBLa-A were used as total genetic material in the study. Blast resistance assay was initially carried out in a greenhouse and further selection was conducted in the endemic blast area, Sukabumi. The selected Molecular marker was STS (Sequence Taq Sites) marker for foreground selection and 384-SNPs (Single Nucleotide Polymorphism) chip for background selection. The number of lines from each cross was selected by greenhouse assay i.e: 25 lines derived from Situ Patenggang/IRBLta and Situ Patenggang/IRBLkp, 21 plants for crossing Situ Patenggang/IRBLi, and 22 plants for Situ Patenggang/IRBLa-A. The results of field experiment revealed that blast resistance response of the selected lines was varied from 0 to 5. Lines of foreground were successfully selected using STS markers specified for Pii, Pita, Pikp and Pia genes. As for the background selection by SNPs markers, some lines carried the recurrent parent genetic background, Situ Patenggang. Overall, 20 resistance lines that harbored the target genes and Situ Patenggang background were obtained. Further observation was entailed to these twenty selected lines in order to attain promising lines candidate for blast resistance.

Response to Cassava Brown Streak Disease Infections in Local and Improved Cassava Genotypes under Field and Greenhouse Assays in Lower Eastern Kenya

Cassava brown streak disease (CBSD) is caused by two cassava brown streak viruses (CBSVs) transmitted by whiteflies (Bemisia tabaci). CBSD significantly inhibits cassava production in Kenya through losses of up to 100% in farmer-preferred but susceptible varieties. As a management strategy, the present study evaluated the effect of CBSD on two local varieties (Thika-5 & Serere) and 15 improved genotypes in lower Eastern Kenya. Between October 2016 and June 2017, the genotypes were infected with CBSVs through whitefly transmission under field experiment at SEKU research farm (1.31ºS, 37.75ºE) and chip-bud grafting at KALRO-Katumani (1.35ºS, 37.14ºS) greenhouse conditions. RCBD and CRD experimental designs were respectively applied in field and greenhouse assays. CBSD symptoms were quantified through disease incidence (DIC) and severity (DSY) every 3 months for the field experiment and weekly for greenhouse assay. At harvest, storage root necrosis (SRN) was scored and non-necrotic roots weighed as marketable root yield (MRY). Molecular diagnostics was accomplished through duplex RT-PCR. Results revealed significantly (P≤0.01) higher foliar field DIC (81- 100%) and SRN (2.3 – 5.0) recorded in Thika-5 and Serere compared to all the improved genotypes that were foliarly asymptomatic (0% DIC and mean SRN of 1.0). Concomitantly and substantially lower (P≤0.01) MRY (1.99 – 2.16 t/ha) were bulked by Thika-5 and Serere compared to 10 improved genotypes that bulked 5.81 – 9.21 t/ha MRY. Upon chip-bud graft infection, Thika-5 and Serere showed higher DIC of 81 – 90% compared to four improved genotypes with 20 - 35% DIC. Correlations between MRY, DIC and SRN were inverse and significant (P≤0.01). RT-PCR detected pre-dominantly CBSV. In conclusion, the natural whitefly-based transmission of CBSVs was ineffective compared to chip-bud grafting. The inverse correlations between CBSD symptoms and yield corroborated the deleterious impact of CBSD on cassava production. The ten improved, high yielding and asymptomatic genotypes identified in the current study could potentially be used to confer resistance against CBSD into farmer-preferred but often sensitive varieties.

Symbiosis of selected Rhizobium leguminosarum bv. viciae strains with diverse pea genotypes: effects on biological nitrogen fixation

Biological nitrogen fixation (BNF) can be improved by optimizing the interaction between the rhizobial inoculant and pea (Pisum sativum L.), leading to increased productivity and reduced nitrogen (N) fertilizer use. Eight Rhizobium leguminosarum bv. viciae strains were used to inoculate the super-nodulating pea mutant Rondo-nod3 (fix+), the hyper-nodulating pea mutant Frisson P88 Sym29, CDC Meadow commercial control, and the non-nodulating mutant Frisson P56 (nod–) to evaluate BNF in a greenhouse assay. Significant differences in strain × cultivar interactions were detected for shoot and root dry masses, which ranged from 1.8 to 4.7 g and from 0.27 to 0.73 g per plant, respectively; for nodule number on lateral roots, which ranged from 25 to 430 per plant; for amount of fixed N2, which ranged from 15 to 67 mg and from 4 to 15 mg per plant for shoot and root tissues, respectively; and for percentage of N derived from atmosphere (%Ndfa), which ranged from 37% to 61% and from 35% to 65% for shoot and root tissue, respectively. Strain × cultivar interactions in this study could contribute to identification of superior strains and pea breeding lines with genetic superiority in BNF. Nodule production in pea plants was not necessarily correlated with the amount of fixed N2, suggesting nodule activity is more important to BNF than is nodule number.