Scientific paper

# The Title of the Virus in the Inoculum Affects the Title of the Viral RNA in the Host Plant and the Occurrence of the Disease Symptoms

Maruša Pompe-Novak,* Maja Križnik and Kristina Gruden

National Institute of Biology, Ljubljana, Slovenia

* Corresponding author: E-mail: marusa.pompe.novak@nib.si

Received: 07-04-2018

Dedicated to the memory of Prof. Dr. Igor Kregar

## Abstract

*Potato virus Y (PVY) is the most economically important potato virus, therefore extensive research is focusing on eluci- dation of its interaction with the host. To obtain repeatable results, strict standardization of research methods is crucial. Mechanical inoculation by rubbing sap from a PVY infected plant onto the leaf surface together with a fine abrasive powder is the most convenient way of experimental transmission of PVY to host plants. However, factors determining reproducibility of this process need to be determined. In the present study, it was shown that higher titre of the virus in the inoculum resulted in faster increase of PVYNTN RNA titre in the inoculated leaves, as well as in faster translocation of PVYNTN from inoculated leaves into upper non-inoculated leaves. The final titre of PVYNTN RNA in upper non-inocu- lated leaves was independent of the virus titre in the inoculum. In addition, the occurrence of the disease symptoms was followed and the dependence to the titre of the virus in the inoculum was observed.*

**Keywords:** Potato virus Y; PVY; potato; mechanical inoculation; inoculum; virus titre; symptoms

## 1. Introduction

*Potato virus Y (PVY) is the most economically im- portant potato virus affecting potato production world- wide¹ and it was classified as one of ten most important plant viruses overall.² In sensitive potato cultivars, it can cause potato tuber necrosis ring spot disease.³ On the green part of a plant, local lesions, chlorosis, mosaic, crinkling, systemic necrosis, leaf drop and plant death can appear.⁴ The outcome of the interaction depends on genotypes of both, potato host plant and PVY pathogen. In addition, the response of a potato towards PVY infection can vary with various environmental conditions and various develop- mental and physiological growth stages of a plant.⁵

PVY infected potato plants can exhibit either a com- patible or an incompatible response.⁶ Plants exhibiting a compatible interaction are susceptible and the virus can replicate and invade the plants.⁷ Susceptible potato plants can be either sensitive or tolerant to PVY infection. Sensi- tive potato plants develop disease symptoms, while toler- ant plants develop no or very mild symptoms, although they can accumulate high titre of the virus.⁸

In an incompatible interaction, plants resist the virus by restricting cell invasion, virus replication and/or virus spread. Plants can respond to virus infection with an ex- treme resistance (ER) or a hypersensitive response (HR). In the case of ER, potato plants show no symptoms or very limited necrosis in the form of pinpoint lesions.⁹ Virus ti- tres remain extremely low, below the limit of detection also in the initially infected leaf.⁹,¹⁰,¹¹,¹² In the case of HR, virus translocation from the initially infected leaf to other parts of the plant is prevented, although the virus replication and initial cell-to-cell movement are not blocked. Later, most of the infected cells die, which result in a localised necrotic lesion at the site of infection.¹²,¹³

In the natural environment, PVY is transmitted by sap-feeding aphid vector, or vegetatively through propa- gated potato tubers. Experimentally, PVY can be transmit- ted by mechanical means such as grafting or rubbing of a sap from a PVY infected plant onto the leaf surface togeth- er with a fine abrasive powder, e.g. carborundum.¹⁴ The latter method is a convenient mean for experimental transmission of the PVY to host plants, although strict standardization of the method is crucial for repeatable re-
sults. The aim of the present study was to investigate the relation between the titre of the virus in the inoculum and the titre of the viral RNA in the host plant, and additionally to determine whether there is the correlation between PVY titre in the inoculum and induction of disease symptom development in inoculated plants.

## 2. Experimental

96 virus free potato plants (*Solanum tuberosum* L.) of cultivar Désireé and 105 virus free plants of cv. Igor from stem node tissue culture (cultivated for 2 weeks) were planted in soil and kept at 21 ± 2 °C in growth chambers, with illumination at 70 µMm⁻²s⁻¹ (Osram L36W/77 lamp), a photoperiod of 16 h and relative humidity 70%. After 4 weeks, three bottom leaves of each plant were mechanically inoculated by rubbing the inoculum onto the leaf surface together with a fine abrasive powder of carborundum (0.037 mm). Inoculum was prepared by grinding PVYNTN (NIB-NTN isolate, AJ585342, referred also as NTN-Slo Isolate¹⁵) infected plants of cv. Pentland Squire from node tissue culture in the grinding buffer (0.02 M phosphate buffer, 0.01 M DETC, pH 7.6) in the ratios (w/v) 1:5, 1:10, 1:50 and 1:100. For control mock inoculation, healthy plants of cv. Pentland Squire from node tissue culture were ground in the grinding buffer in the ratio (w/v) 1:5. After 10 min incubation, the inoculum was washed from the leaf surface by tap water.

On potato plants of cv. Igor disease symptom development was monitored at 0, 3, 4, 5, 6, 7, 10 and 14 days post inoculation (dpi). At the same time points, samples from three plants per treatment were collected for the quantification of PVY RNA. For that purpose, the right distal quarter of the second inoculated leaf and the right distal quarter of the second upper non-inoculated leaf were collected and further analysed separately from each plant of cv. Désireé. Collected leaf tissues were frozen immediately in liquid nitrogen and stored at -80 °C for further analysis.

Plant tissue was homogenised by Tissue Lyser (Qiagen, Hilden, Germany). RNA was isolated by MagMAX™ Plant RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) including DNAse treatment. RNA concentration, quality, and purity were assessed using agarose gel electrophoresis and NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). RT-qPCR reactions were performed on 2 µl of extracted RNA in a total volume of 10 µl using the AgPath-IDTM One-Step RT-PCR Kit

(Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.

Primer and probe concentrations, together with PCR amplification conditions, were as in Kogovšek *et al.*¹⁶ Two dilutions of RNA were analysed, each in duplicate. Reactions were carried out using the ABI 7900HT Sequence Detection System (Applied Biosystems, Foster

City, CA, USA). RT-qPCR conditions included a reverse transcription step (10 min at 48 °C) followed by 40 amplification cycles (1 min at 60 °C, 15 s at 95 °C). Data were analysed using the SDS v 2.3 software (ABI, Foster City, CA, USA). PVY RNA amount was quantified using a relative standard curve method by normalization to the endogenous control cox mRNA by quantGenius.¹⁷ The significance of differences (p ≤ 0.05) between mean values was determined by the Student's t-test.

## 3. Results and Discussion

The titre of PVYNTN RNA was studied in potato host plants of tolerant cv. Désireé after inoculation with the inocula obtained by grinding PVYNTN infected plant tissue in grinding buffer in different ratios (w/v), 1:5, 1:10 and 1:100, and therefore containing different titres of PVYNTN. The amount of PVYNTN RNA detected immediately after inoculation (0 dpi) correlated with the titre of PVYNTN in the inoculum, indicating that the remnants of PVYNTN RNA on the leaf surface after washing the inoculum was detected.

From 0 dpi to 4 dpi the detected amount of PVYNTN RNA decreased (Figure 1) due to degradation of PVYNTN RNA on the leaf surface. It was already demonstrated in previous studies¹⁸ that it is possible to detect the traces of the inoculum on the leaf surface of extremely resistant cv. Santé (in which PVY cannot multiply) even at 14 dpi; and to detect viral RNA in dry inoculum 12 days after its preparation. Double-stranded RNA products shown to arise during viral replication of *Potyviridae*¹⁹,²⁰ can explain the stability of viral RNA.

It is hypothesised that PVY enters the initial potato cells for further multiplication through broken leaf hairs

<figure><img src="image_1.png" /><figcaption>**Figure 1.** The relative amount of PVYNTN RNA measured by RT-qPCR in the inoculated leaves of cv. Désireé from 0 to 7 dpi. Error bars represent the standard error (n = 3). Statistical comparison was made between plants inoculated with the inoculum in which PVYNTN infected potato sap was diluted in buffer in the ratio 1:100 and plants inoculated with the inocula in which PVYNTN infected potato sap was diluted in buffer in the ratios 1:5 or 1:10. Statistically significant differences (p ≤ 0.05) are indicated with.*</figcaption></figure>
(broken by rubbing at mechanical inoculation). At 5 dpi the amount of newly multiplied PVY<sup>NTN</sup> RNA exceeded the amount of PVY<sup>NTN</sup> RNA in the remnants of the inoculum on the leaf surface of the plants inoculated with the inocula in which PVY<sup>NTN</sup> infected potato sap was diluted in buffer in the ratios 1:5 or 1:10 (Figure 1). From 5 to 7 dpi, higher PVY<sup>NTN</sup> RNA titres were observed in plants inoculated with more concentrated inoculum (Figure 1). In leaves, inoculated with the inoculum in which PVY<sup>NTN</sup> infected potato sap was diluted in buffer in the ratio 1:100, the titre of PVY<sup>NTN</sup> probably increased later than 7 dpi, as also the translocation of PVY<sup>NTN</sup> from inoculated into upper noninoculated leaves occurred later (Figure 2).

In inoculated leaves of potato the concentrical distri-
bution of PVY around the point of infection was shown
during HR.¹² In inoculated leaves of tobacco (Nicotiana
tabacum L.) cv. Xanthi movement of PVY from initially
inoculated cells to neighbour cells in concentrical circles,
loading of PVY into the veins and translocation of PVY
into upper non-inoculated leaves was shown in tolerant
interaction.²¹ Our results in tolerant potato-PVY interac-
tion showed that the translocation of PVYNTN from inocu-
lated into upper non-inoculated leaves of potato cv. Dé-
sireé occurred regardless of the virus titre in the inoculum,
while the speed of the process was dependent on the virus
titre in the inoculum (Figure 2). Although the multiplica-
tion of PVYNTN in inoculated leaves and the translocation
of PVYNTN from inoculated into upper non-inoculated
leaves was slower in the case of lower viral titre in the inoc-
ulum, the final titre of PVYNTN RNA in upper non-inocu-
lated leaves 14 dpi was independent of the virus titre in the
inoculum (Figure 2). Even much longer times (a few
months) are required for the spread of PVYNTN from nu-
trient solution containing extremely low titre of PVYNTN,
through the roots, to the green parts of the plants as shown
in experiments using a hydroponic system.²²

Additionally, the disease symptom development af-
ter inoculation with different PVYNTN titre was monitored
on potato plants of cv. Igor, which is one of the most sus-
ceptible and sensitive potato varieties to PVYNTN. A few
days after infection, local lesions appeared on inoculated
leaves, followed by leaf chlorosis (yellowing of the leaves)
and leaf drop of inoculated leaves. In general, the severity
of the disease symptoms correlated with the titre of PVY-
NTN in the inoculum. The appearance and the development
of disease symptoms was slower in the plants inoculated
with lower titre of PVYNTN in the inoculum (Figure 3),
what is shown also by the correlation between the titre of
PVYNTN in the inoculum and the leaf drop (Figure 4). By
14 dpi, all 3 inoculated leaves dropped in plants inoculated
with the inocula diluted in the ratios 1:5 or 1:10. At the
same time, in plants inoculated with the inocula diluted in
the ratios 1:50 or 1:100 only the 1st and the 2nd inoculated
leaf dropped, while the 3rd inoculated leaf showed more
severe symptoms in plants inoculated with the inoculum
diluted in the ratio 1:50 as compared to the plants inocu-
lated with the inoculum diluted in the ratio 1:100 (Figure
3). No symptoms appeared on control potato plants, where
also no leaf drop was observed at 14 dpi (Figure 3, right
panel).

Besides the high titre of PVYNTN in the inoculum,
also too much carborudum or too excessive rubbing at
mechanical inoculation promotes the leaf drop on inocu-
lated plants (data not shown). Early leaf drop of inoculated
leaves can prevent the spread of the virus to upper non-in-
oculated leaves and the development of symptoms in up-
per non-inoculated leaves. In PVYNTN - cv. Igor interac-
tion, the absence of symptoms in upper non-inoculated
leaves indicates with a high probability the absence of the
virus in upper non-inoculated leaves.

Although the plants are kept in the same growth
chamber controlled environment with the same tempera-

<figure><img src="image_2.png" /><figcaption>**Figure 2.** The relative amount of PVYNTN RNA measured by RT-qPCR in upper non-inoculated leaves of cv. Désireé from 6 to 14 dpi. Error bars represent the standard error (n = 3). Statistical comparison was made between plants inoculated with the inoculum in which PVYNTN infected potato sap was diluted in buffer in the ratio 1:100 and plants inoculated with the inocula in which PVYNTN infected potato sap was diluted in buffer in the ratios 1:5 or 1:10. None of the differences was statistically significant (p ≤ 0.05).</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 3.** Disease symptoms on potato plants of cv. Igor at 14 days after inoculation with the inocula in which PVY<sup>NTN</sup> infected potato sap was diluted in buffer in the ratios 1:5 (left), 1:10 (second from the left), 1:50 (middle) or 1:100 (second from the right); and control potato plants (right).</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 4.** The average number of dropped inoculated leaves per plant in cv. Igor after infection with PVY<sup>NTN</sup> from 0 dpi to 14 dpi. Error bars represent the standard error (n = 3). Statistical comparison was made between plants inoculated with the inoculum in which PVY<sup>NTN</sup> infected potato sap was diluted in buffer in the ratio 1:100 and plants inoculated with the inocula in which PVY<sup>NTN</sup> infected potato sap was diluted in buffer in the ratios 1:50, 1:10 or 1:5. Statistically significant differences (p ≤ 0.05) are indicated with.*</figcaption></figure>

ture, relative humidity, illumination and photoperiod, the plant's response to viral infection (including the development of symptoms) can vary between experiments (biological repetitions) due to yet unknown parameters (e.g. physiological state of the plant, season...).

## 4. Conclusions

We can conclude that higher titre of PVY<sup>NTN</sup> in the inoculum resulted in faster increase of PVY<sup>NTN</sup> RNA titre in the inoculated leaves and in faster translocation of PVY<sup>NTN</sup> from inoculated leaves into upper non-inoculated leaves. The final amount of PVY<sup>NTN</sup> RNA in upper noninoculated leaves was independent of PVY<sup>NTN</sup> titre in the inoculum. In addition, the occurrence of the disease symptoms was slower in the plants inoculated with lower titre of PVY<sup>NTN</sup> in the inoculum as compared to the plants inoculated with higher titre of PVY<sup>NTN</sup> in the inoculum. Thus to obtain reproducible results of any biochemical, physiological or molecular study of potato - PVY interaction one should use standardised titre of the virus in the inoculum.

## 5. Acknowledgment

The research was financially supported by the Slovenian Research Agency (research core funding No. P4-0165). The authors thank Tjaša Cof, Mihaela Leupušček and Živa Marinko, who joined to the research as students; and Neža Turnšek for technical help.

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## Povzetek

Virus krompirja Y (*Potato virus Y*, PVY) je ekonomsko najpomembnejši virus krompirja, zato se številne raziskave osredotočajo na študij interakcije PVY z gostiteljsko rastlino. Za zagotavljanje ponovljivosti rezultatov je pomembna standardezacija raziskovalnih metod. Najpogostejši način prenosa PVY na gostiteljsko rastlino v eksperimentalnih pogojih je mehanska inokulacija, pri kateri na površino lista nanesemo abrazivno sredstvo in sok okužene rastline, pri čemer pa je potrebno določiti dejavnike, ki so ključni za ponovljivost tega postopka. V raziskavi smo pokazali, da se je višji titer virusa v inokulumu odražal v hitrejšem dvigu titra RNA PVYNTN v inokuliranih listih ter v hitrejšem širjenju PVYNTN iz inukuliranih listov v zgornje neinokulirane liste. Končni titer RNA PVYNTN v zgornjih neinokuliranih listih pa ni bil odvisen od titra virusa v inokulumu. Poleg tega smo opazili tudi, da je bila hitrost pojava bolezenskih znamenj odvisna od titra virusa v inokulumu.