Exploring the phylogenetic diversity of Botryosphaeriaceae and Diaporthe species causing dieback and shoot blight of blueberry in Serbia.

Identifying the precise pathogens responsible for specific plant diseases is imperative for implementing targeted and efficient interventions and mitigating their spread. Dieback and shoot blight significantly diminish the lifespan and productivity of blueberries, yet the causative agents remain largely unidentified. To determine the identity and prevalence of the causal agents of branch dieback and shoot blight, we conducted multi-year and multi-site sampling of diseased highbush blueberries (Vaccinium corymbosum cv. Duke) in Serbia. Sixty-nine monosporic isolates were collected and characterized based on morphological, physiological features and multi-locus phylogenetic analysis of internal transcribed spacer (ITS), ß-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1) sequence data. Five species were identified as causal agents: Diaporthe eres (36 isolates), D. foeniculina (3 isolates), Neopestalotiopsis vaccinii (9 isolates), N. rosae (6 isolates) and Neofusicoccum parvum (15 isolates). The results of the pathogenicity tests performed with the 23 representative isolates confirmed the role of these species as primary pathogens in causing dieback and shoot blight of blueberry, with N. parvum being the most aggressive and D. eres the least. Our study underscores the diversity of genera and species of ascomycetes capable of causing blueberry dieback and shoot blight. Furthermore, our findings indicate that the agents responsible for the disease in Serbia differ from those identified in other regions worldwide.

Efficiency of Biological Typing Methods in Maize Hybrid Genetic Purity Estimation.

A high level of genetic purity in crop varieties must be achieved and maintained for agronomic performance, encouraging investment and innovation in plant breeding and ensuring that the improvements in productivity and quality imparted by breeders are delivered to the consumer. Since the success of hybrid seed production is dependent upon the genetic purity of the parental lines, in this study, the experimental F1exp maize hybrid and its parental inbreeds were used as a model system to examine the discriminative power of morphological, biochemical and SSR markers for seed purity assay. The highest number of off-type plants was estimated by morphological markers. According to the comparison of prolamins and albumins banding patterns of parental and derived F1exp seeds, genetic impurities could not be detected. Molecular analysis detected two types of genetic profile irregularity. Beside its use for verifying varieties of maize, report on umc1545 primer pair ability to detect non-specific bands (i.e., off-types), in both the maternal component and F1exp, which is the first report on this issue yet, strongly supports the recommendation of this SSR marker use for more accurate and time-efficient maize hybrids and parental lines genetic pyrity testing.

Alteration of Metabolites Accumulation in Maize Inbreds Leaf Tissue under Long-Term Water Deficit.

Plants reconfigure their metabolic pathways to cope with water deficit. The aim of this study was to determine the status of the physiological parameters and the content of phenolic acids in the upper most ear leaf of maize inbred lines contrasting in drought tolerance in terms of improved plant productivity e.g., increased grain yield. The experiment was conducted under irrigation and rain-fed conditions. In drought-tolerant lines, the effect of water deficit was reflected through a chlorophyll and nitrogen balance index increase followed by a flavonols index decrease. The opposite trend was noticed in drought susceptible inbreds, with the exception of the anthocyanins index. Moreover, in comparison to irrigation treatment, opposite trends in the correlations between grain yield and physiological parameters found under water deficit conditions indicated the activation of different metabolic pathways in defense against water deficit stress. Concerning phenolic acid content, water deficit caused the reduction of protocatechuic, caffeic, and sinapic acid in all inbreds evaluated. However, the highly pronounced increase of ferulic and especially cinnamic acid content under water deficit conditions indicated possible crucial role of these secondary metabolites in preventing the harmful effects of water deficit stress, which, in turn, might be useful in maize breeding selection for drought tolerance.

Penicillium and Talaromyces Species as Postharvest Pathogens of Pear Fruit (Pyrus communis) in Serbia.

Pears are one of the oldest and the third most important fruit species grown in temperate regions. They are consumed because of their nutritional and health benefits, in fresh form or as various processed products. This article resolves the etiology of the Penicillium-like mold symptoms on pear fruits in Serbia. Samples of pear fruits with blue mold and other Penicillium-like mold symptoms were collected in Serbia from 2016 to 2019, from four storages. The recovered isolates were identified and characterized according to a polyphasic approach. Morphological and physiological analyses were performed on three media and five temperatures, respectively. Four loci (internal transcribed spacer, beta-tubulin, calmodulin, and DNA-dependent RNA polymerase II second largest subunit) were used for sequencing, genetic identification, and phylogenetic analyses. The results of the identification by conventional and molecular methods were in agreement, and they revealed that the obtained isolates belong to five species: Penicillium crustosum, P. expansum, P. italicum, Talaromyces minioluteus, and T. rugulosus. In a pathogenicity test, P. crustosum, P. expansum, T. minioluteus, and T. rugulosus produced decay on artificially inoculated pear fruits, and P. italicum induced tissue response lesions. The results of this study are the first reports of T. minioluteus and T. rugulosus as postharvest pear pathogens. Also, these are the first world records of T. minioluteus, T. rugulosus, and P. italicum on fruits of European pear. Furthermore, this is the first finding of P. crustosum, P. expansum, P. italicum, T. minioluteus, and T. rugulosus on pear fruit in Serbia.

First report of Penicillium olsonii causing postharvest fruit rot on tomato in Serbia.

Tomato (Solanum lycopersicum, L.) is one of the most important vegetable crop in Serbia, with a total production of 111,639 t in 2019 (Statistical Office of the Republic of Serbia). In July 2020, six tomatoes (cv. Balkan) with symptoms of fruit rot were collected from market in Belgrade, Serbia. The incidence of disease was about 2%, and the symptomatic samples were stored for 10 days after harvest. The initial symptoms on fruits were small circular, slightly sunken and water-soaked spots with white mycelia, that progressively expanded into larger grey lesions following the occurrence of sporulation. Isolations were conducted from one spot/fruit. Small pieces (2 to 3 mm2) from the margins of lesions were surface sterilized for 1 min in 1% NaOCl, washed twice with sterile distilled water, and cultivated on potato dextrose agar (PDA) at 25°C. The isolation frequency of Penicillium-like colonies was 100%. In total, six monosporic isolates were obtained and two isolates (SZ-20-6 and SZ-20-7) were selected as representative for morphological and molecular identification, and pathogenicity test. Morphological characteristics of both isolates were observed after growth on malt extract agar (MEA) for 7 days at 25ºC. On MEA, mycelia were white and colonies turned greyish-green with abundant sporulation. On the reverse sides colonies were pale yellow. The mean colony diameter on MEA for isolate SZ-20-6 was 25 ± 1.2 mm and 26 ± 1.0 mm for isolate SZ-20-7. The colony texture was velvety, without exudates and pigmentation. The conidiophores of both isolates were terverticillate, unbranched; phialides were flask shaped with a short neck, and conidia were smooth, greenish and subglobose to ellipsoidal. The conidial diameter for isolate SZ-20-6 was 3 to 4 × 2.5 to 3 µm, and for isolate SZ-20-7 was 3.5 to 4 × 2.5 to 3.5 µm (n =50). Based on these characteristics, isolates were identified as Penicillium olsonii (Pitt 1979). To confirm the morphological identification, genomic DNA was extracted from isolates (SZ-20-6 and SZ-20-7), and the rDNA ITS region and partial ß-tubulin gene (BenA) were amplified using the primers ITS1/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. All sequences showed 99 to 100% similarity to P. olsonii and were deposited in GenBank (ITS, MW130235 and MW130236; BenA, MW145147 and MW145148). In multilocus phylogenetic analysis (ITS+BenA), isolates from this study clustered together with other P. olsonii sequences with 100% bootstrap support. To complete Koch's postulates, asymptomatic fruits of tomato cv. Balkan (five fruits per isolate) were superficially sterilized with 70% ethanol, wounded with a sterile needle and inoculated with 10 µl of a spore suspension (1 × 106 spores/ml). Five control fruits were inoculated with 10 µl of sterile distilled water. The experiment was repeated twice. After 7 days of incubation in a moisture chamber at 25°C, typical grey lesions developed on inoculated fruits. The control fruits remained symptomless. The isolates recovered from symptomatic fruits showed the same morphological features as the original isolates. P. olsonii was previously reported on tomato fruit only in Canada (Chatterton et al. 2012) and Pakistan (Anjum et al. 2018). To our knowledge, this is the first report of P. olsonii causing postharvest fruit rot on tomato in Serbia, and in Europe, as well. Therefore, it is essential to monitor spreading of P. olsonii on tomato and other crops in storages, and develop efficient disease management strategies. References: Anjum, N. et al. 2018. Plant Dis. 102:451. Chatterton, S., et al. 2012. Can. J. Plant Pathol. 34:524. Glass, N. L. and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. Pitt, J. I. 1979. The Genus Penicillium and its Teleomorophic States Eupenicillium and Talaromyces. Academic Press, London, U.K. Statistical Office of the Republic of Serbia. https://www.stat.gov.rs/en-US/ White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Funding: This research was financed by the Ministry of Education, Science and Technical Development of the Republic of Serbia, grant 451-03-68/2020-14/200010.

Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli.

Bacterial fruit blotch and seedling blight, caused by Acidovorax citrulli, is one of the most destructive diseases of melon and watermelon in many countries. Pathogen-free seed and cultural practices are major pillars of the disease control. However, use of bacteriophages as natural biocontrol agents might also contribute to the disease management. Therefore, we isolated 12 bacteriophages specific to A. citrulli, from phyllosphere and rhizosphere of diseased watermelon plants. The phage strains were characterized based on their host range, plaque and virion morphology, thermal inactivation point, adsorption rate, one step growth curve, restriction fragment length polymorphism (RFLP), and genomic analysis. Transmission electron microscopy of three phage strains indicated that they belong to the order Caudovirales, family Siphoviridae. All phages lysed 30 out of 32 tested A. citrulli strains isolated in Serbia, and did not lyse other less related bacterial species. They produced clear plaques, 2 mm in diameter, on bacterial lawns of different A. citrulli strains after 24 h of incubation. The thermal inactivation point was 66 or 67°C. They were stable at pH 5-9, but were sensitive to chloroform and inactivated in either 5 or 10 min exposure to ultraviolet (UV) light. RFLP analysis using EcoRI, BsmI and BamHI enzymes did not show genetic differences among the tested phages. Adsorption rate and one step growth curve were determined for the Acidovorax phage ACF1. Draft genome sequence of the ACF1 phage was 59.377 bp in size, with guanine-cytosine (GC) content 64.5%, including 89 open reading frames. This phage shared a very high genomic identity with Acidovorax phage ACPWH, isolated in South Korea. Evaluation of systemic nature of ACF1 strain showed that it can be absorbed by roots and translocated to upper parts of watermelon plants where it survived up to 10 days.

Talaromyces minioluteus: New Postharvest Fungal Pathogen in Serbia.

Talaromyces minioluteus is one of the important species of genus Talaromyces, which has cosmopolitan distribution and is encountered on a wide range of different habitats. This species has not been considered as an important plant pathogen, even though it has been isolated from various plant hosts. Fruits and vegetables with Penicillium-like mold symptoms were collected from 2015 to 2017 from markets in Serbia. Isolates originating from quince, tomato, and orange fruits, onion bulbs, and potato tubers were identified and characterized on a morphological, physiological, and molecular level. Morphological and physiological examination included observing micromorphology, testing growth on six different media and at five different temperatures, and production of three enzymes. Molecular identification and characterization were performed using four molecular markers: internal transcribed spacer, ß-tubulin, calmodulin, and DNA-dependent RNA polymerase II second largest subunit. The results of morphological and molecular analyses were in agreement, and they proved that the obtained isolates are T. minioluteus. In the pathogenicity assay, T. minioluteus was confirmed as a pathogen of all species tested with the exception of potato tubers. This is the first report of T. minioluteus as a postharvest plant pathogen on quince, tomato, and orange fruit and onion bulbs. Also, this is the first record of T. minioluteus in Serbia.

Characterization of Gnomoniopsis idaeicola, the Causal Agent of Canker and Wilting of Blackberry in Serbia.

Blackberry cane diseases with the symptoms of necrosis, canker, and wilting are caused by several fungi worldwide. Surveys conducted from 2013 to 2016 in Serbia revealed the occurrence of Gnomoniopsis idaeicola, the causal agent of cane canker and wilting, which was found to be distributed in almost half of the surveyed orchards, in three blackberry cultivars, and with disease incidence of up to 80%. Wide distribution and high disease incidence suggest that G. idaeicola has been present in Serbia for some time. Out of 427 samples, a total of 65 G. idaeicola isolates were obtained (isolation rate of 34.19%). Reference isolates, originating from different localities, were conventionally and molecularly identified and characterized. G. idaeicola was detected in single and mixed infections with fungi from genera Paraconiothyrium, Colletotrichum, Diaporthe, Botryosphaeria, Botrytis, Septoria, Neofusicoccum, and Discostroma, and no diagnostically specific symptoms could be related directly to the G. idaeicola infection. In orchards solely infected with G. idaeicola, blackberry plant mortality was up to 40%, and yield loses were estimated at 50%. G. idaeicola isolates included in this study demonstrated intraspecies diversity in morphological, biological, pathogenic, and molecular features, which indicates that population in Serbia may be of different origin. This is the first record of a massive outbreak of G. idaeicola infection, illustrating its capability of harmful influence on blackberry production. This study represents the initial step in studying G. idaeicola as a new blackberry pathogen in Serbia, aiming at developing efficient control measures.

Development of High Tryptophan Maize Near Isogenic Lines Adapted to Temperate Regions through Marker Assisted Selection - Impediments and Benefits.

Breeding program aimed at converting standard maize inbred lines to their quality protein maize (QPM) counterparts for growing in temperate climate is being conducted at Maize Research Institute (MRI). The objective of the research presented herein was to develop QPM versions of two commercial ZP inbreds through marker assisted selection (MAS) with opaque2 specific molecular markers, while maintaining their good agronomic performances and combining abilities. Donor line was a tropical QPM line CML 144. After two backcross and three selfing generations, six near isogenic lines (NILs) with 93% recovery of the recurrent parent genome were created from one cross. Average increments of 30% in tryptophan content and 36% in quality index were obtained, as well as kernels with less than 25% opaque endosperm. Grain yield was increased by 11-31% and combining abilities of the improved lines were on a par with the original line. Correlations between biochemical and agronomic parameters revealed that selection for plant height, ear length and kernel row number together with tryptophan content could be recommended for development of QPM with this material. However, several impediments emerged during selection. Major drawbacks in NIL development were small number of opaque2 recessive homozygotes (4.5% and 7.6% in BC2F2 of two crosses) and poor seed set throughout selection, which led to the loss of one cross. Moreover, in the other cross many plants in different generations had to be omitted from further selection due to the insufficient number of kernels. This phenomenon could be explained by incompatibility between pollen and style, possibly due to the exotic donor germplasm. Overall, it could be expected that the use of NILs, which are adapted to temperate climate and have high percentage of domestic germplasm, would outbalance the noted impediments and increase MAS efficiency in different breeding programs.

In vitro and in vivo antifungal properties of cysteine proteinase inhibitor from green kiwifruit.

BACKGROUND: Higher plants possess several mechanisms of defense against plant pathogens. Proteins actively synthesized in response to those stresses are called defense-related proteins which, among others, include certain protease inhibitors. It is of particular relevance to investigate plant natural defense mechanisms for pathogen control which include cystatins-specific inhibitors of cysteine proteases. RESULTS: In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. Immuno-tissue print results indicated that CPI is most abundant in the outer layer of pericarp, near the peel, and the inner most part of the pulp-sites where it could act as a natural barrier against pathogens entering the fruit. The purified protein (15 µmol L(-1)) showed antifungal activity against two phytopathogenic fungi (Alternaria radicina and Botrytis cinerea) by inhibiting fungal spore germination. In vivo, CPI (10 µmol L(-1)) was able to prevent artificial infection of apple and carrot with spore suspension of B. cinerea and A. radicina, respectively. It also exerted activity on both intracellular and fermentation fluid proteinases. CONCLUSION: Identification and characterization of plant defense molecules is the first step towards creation of improved methods for pathogen control based on naturally occurring molecules.